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Effects of local anesthesia on substance P and CGRP content of the human dental pulp
0099-2399/97/2307-0416503.00/0 JOURNALOF ENDODONTlCS Copyright © 1997 by The American Association of Endodontists
Printed in U.S.A. VOL. 23, NO. 7, JULY 1997
Effects of Local Anesthesia on Substance P and CGRP Content of the Human Dental Pulp C. Pertl, R. Amann, E. Odell, P. D. Robinson, and S. Kim
The aim of the study was to determine immunoreactive Substance P (iSP) and immunoreactive calcitonin gene-related Peptide (iCGRP) content in the human dental pulp and whether local anesthesia has an effect on the neuropeptide content. Dental pulps were obtained from patients, who underwent surgical extraction of all 4 impacted wisdom teeth under general isoflurane gas anesthesia. There was a very high interindividual variation in tissue content, with small variation in levels found in teeth from the same patient. Pulps obtained from lower teeth without local anesthesia contained an average of 131 +_ 62 fmol/mg protein of iCGRP and 15 --- 9 fmol/mg iSP (n = 10). With additional mandibular block anesthesia the values were 194 _ 71 fmol/mg iCGRP (statistically significant, p = 0.0356, Mann-Whitney-Rank-Sum-Test) and 12 _+ 3.6 fmol/mg iSP. The results suggest that local anesthesia attenuates neuropeptide release in the human dental pulp during surgical extraction.
iCGRP using radioimmunoassays. Both pulpal exposure and acid etch produced significant decreases in tissue levels of iSP and iCGRP. Three to 10 days after injury iSP levels decreased to about 10% of baseline values, while iCGRP levels decreased to about 45% of baseline measures. With the method of in vitro superfusion of bovine dental pulp, Hargreaves et al. (9) found a 17-fold greater concentration of iCGRP than iSP. Administration of potassium chloride (50 mM) increased levels of iCGRP 3- to 5 times in the superfusate. It has further been shown that potassium-chloride-induced increase in pulpal blood flow was significantly inhibited following blockade of afferent neurons with capsaicin (10). The loss of iCGRP and iSP from electrically stimulated feline teeth pretreated with the sympathetic blocker guanethidine also suggests that these mediators may be responsible for vasodilatation (ll). The human dental pulp is richly innervated by both myelinated and unmyelinated afferent nerves. The subgroup of unmyelinated sensory fibers that contain CGRP and SP forms numerous endings in the coronal pulp and dentin and along many pulpal blood vessels (12, 13). Most of the studies in this field have been carried out in animals and their relevance to humans is unclear. The aims of this study were to determine SP and CGRP content in the human pulp after extraction under general anesthesia and to determine whether additional local anesthesia has an effect on their neuropeptide content.
Because the dental pulp resides in a rigid environment there is a need to control vasodilatation and to limit changes in tissue pressure that might disturb adequate blood perfusion. Changes in microcirculation and haemodynamics are naturally closely related to inflammatory responses. Various stimuli induce vasodilatation and extravasation and consequently increase interstitial pressure in the pulp of animals. It is very likely that these responses are mediated by release of neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) from fine caliber, capsaicin sensitive nerves (1, 2, 3, 4). Several studies by Byers and her colleagues (5, 6, 7) have evaluated the effects of different types of injuries on the immunohistochemical staining of peripheral CGRP fibers in the rat dental pulp. They found distinct neuropeptide responses, mild injuries producing an increase in sprouting of immunoreactive CGRP (iCGRP). Severe pulpal trauma resulted in either a reduction or an elimination of iCGRP staining. Grutzner et al. (8) investigated the effect of injury-induced changes in rat pulpal levels of both immunoreactive SP (iSP) and
METHODS Human dental pulps were obtained from patients who underwent surgical extraction of all 4 wisdom teeth under general isoflurane gas anesthesia. Only those teeth for which the following criteria were fulfilled were included: 1: similar (upright) position of left and right wisdom tooth; 2: full impaction; 3: similar length of operation on left and right tooth (-+5 min); 4: entire duration of the surgical extraction of one tooth (incision-extraction) did not exceed 15 min; and 5: teeth were completely intact after extraction. Local anesthetic with 1 carpule (1.8 ml) of prilocaine 4% without vasoconstrictor was given on the experimental side: buccal and lingual infiltration for the maxillary teeth and mandibular block for the mandibular teeth. The surgical procedure was performed in a conventional manner. Round burs at high speed were used for bone removal and
416
Local Anesthesia and Pulpal NeuropepUdes
Vol. 23, No. 7, July 1997 elevators for tooth extraction. All teeth were put in a perforated plastic tube and were frozen in liquid nitrogen instantly after extraction. During this procedure enamel and dentin fractured due to different thermal expansion of the different tooth tissues allowing easy removal of the coronal pulp in a frozen state. The coronal portions of 48 human wisdom tooth pulps obtained ti'om 14 patients were evaluated. Eight patients were female, six male, and their mean age was 17.7 (rain 15-max 22) years. The pulps were processed for radioimmunoassay and the results were calculated per mg of tissue protein content. For determination of iCGRP and iSP, samples were homogenized and extracted in 2 N acetic acid. An aliquot of the homogenate was used for protein determination (Bio-Rad). After centrifugation, the supernatants were lyophilized and used for radioimmunoassay, iCGRP was determined using antiserum RAS 6009N (Peninsula) and the radioligand (2-[125J]histididyl-10) CGRP (Amersham). iSP was determined using antiserum Rd2 and the radioligand [125J] BoltonHunter-substance P (Amersham). For high pressure liquid chromatography (HPLC), samples were pooled and injected on a reversed-phase nucleosil C18 column (Waters). Elution was performed by an 85-rain gradient from 20 to 60% acetonitrile at a flow rate of 1.5 ml/min. The fractions (1 rain) were lyophilized and assayed for CGRP as described above. Data are shown as means ± S.E.M.; statistical significance was determined using the Mann-Whitney Rank Sum Test (Sigma Stat statistical software, Jandel Scientific, Erkrath, Germany). Informed consent was obtained from all patients, and experiments were approved by the ethics committee of UMDS Guys and St. Thomas's Hospital, London.
RESULTS There was a large interindividual variation of tissue concentrations of iCGRP (10-850 fmol/mg protein) and iSP (1.17-127 fmol/mg protein) found in the control teeth, which had not been anesthetized with prilocaine. The neuropeptide content of different pulps in the same patient was much more constant. The difference in iCGRP or iSP content between different specimens from the same individual did not exceed a factor of 2.3. This was found with the exception of one patient where iCGRP of the upper third molar pulp was 5.2-fold higher than that of the corresponding mandibular. Pulps obtained from lower teeth without local anesthesia contained an average of 131 ± 62 fmol/mg iCGRP and 15 + 9 fmol/mg iSP (n = 10). With mandibular block anesthesia using one carpule prilocaine (4%) the values were 194 ± 71 fmol/mg iCGRP and 12 + 3.6 fmol/mg iSP. In the case of iCGRP the difference between control side and local anesthesia reached statistical significance (p = 0.0356, Mann Whitney Rank Sum Test). The identity of iCGRP was verified by HPLC: more than 90% of immunoreactivity eluted at the position of synthetic human-CGRP. When pulpal neuropeptide content of one side was compared with the contralateral tooth in the same patient, local anesthesia caused a considerable increase (Fig. 1). The neuropeptide content o]" pulps from upper control teeth contained 143 + 59 fmol/mg iCGRP and 9 -+ 3.7 fmol/mg iSP. Corresponding tissue levels of pulps on which side an additional buccal and lingual infiltration of a total of one carpule prilocaine 4% was given were 238 +_ 81 fmol/mg iCGRP and 18 _+ 10 fmol/mg iSP. Although there seemed to be a tendency for increased neuropeptide content after extraction under local anes-
417
300 t~ >
0) 200 +.J cO 0 0
100 Nerve block
Infiltration
FIG. 1. Pulpal neuropeptide content of the side with additional local anesthesia was compared with the contralateral tooth of the same
patient. Mandibular block anesthesia resulted in a iCGRP content of 227% (-+42%) and iSP level of 139% (4-16%) of the control side. The iCGRP and iSP levels under buccal and lingual infiltration were 153% (±20%) and 151% (+-18%) respectively. Statistical significance was calculated using raw data.
thetic infiltration (Fig. l), there was no statistically significant difference.
DISCUSSION In immunohistochemical studies of the human dental pulp more CGRP than SP fibers have been found. Both neuropeptides were found predominantly around pulpal blood vessels but also in the subodontoblastic region (l l, 12). Radioimmunoassay of tissue extracts from bovine dental pulp demonstrated a 17 times higher iCGRP level than iSP (9). We found a similar relationship in the evaluation of human coronal pulp tissue amounting to an approximately 10-fold higher concentration of CGRP than SP. We used a homogeneous group of extracted teeth including only completely intact, impacted third molars from 15 to 22 (mean 17.4) year old patients. Interestingly there was a very high interindividual variation in neuropeptide content. The stability of each individual's specific neuropeptide content is shown by the small variation in levels found in teeth from the same patient. It remains to be determined whether these findings have any physiological relevance such as individual dimension and different capacity of neurovascular mechanisms or different pain thresholds. There seemed to be no correlation in neuropeptide levels between roots with wide open apical foramina and those with more or less formed apices. This observation is in accordance with the immunohistochemical evaluation of Luthman et al. (12), who could not demonstrate any difference in the occurrence and distribution of iCGRP, iSP, and other neurochemical markers in pulps obtained from immature and fully formed teeth. The present study has also demonstrated that dynamic changes in neuropeptide content in the dental pulp occur in humans. The neurovascular reactions induced by noxious stimulation have been studied in numerous animal models. Electrical stimulation, CGRPand SP-infusion, application of capsaicin (the pungent ingredient of red peppers acts as a selective activator of unmyelinated afferent nerves), cavity preparation and percussion cause pulpal vasodilatation and an inflammatory response. Further, it has been shown that the effect of this variety of stimuli depend to a large extent on activation of afferent nerves (1, 3, 4). Application of local anes-
418
Journal of Endodontics
Pertl, et al.
thetic has been shown to block feline pulpal blood-flow increase induced by ultrasonic stimulation and KCI application to a deep dentinal cavity (2, 15). To study the effect of local anesthesia on neuropeptide release during the surgical removal of impacted wisdom teeth, mandibular block anesthesia and maxillary buccal and lingual infiltration were administered on one side. Local anesthesia given to patients in addition to general anesthesia is routinely used. It has been shown that this additional blockade of nociceptive input can decrease pain beyond the immediate postoperative period (16). We found a statistically significantly higher content of iCGRP under mandibular block anesthesia, suggesting that during the trauma of surgical extraction a smaller amount of the neurotransmitter was released, inactivated, and carried off by pulpal blood vessels than in corresponding control teeth. The same tendency was observed with infiltration of prilocaine around upper impacted wisdom teeth but did not reach statistical significance. Whether this is due to the different mechanism or amount of inhibition induced by either local infiltration or nerve block anesthesia or the greater surgical trauma required for extraction of lower impacted wisdom teeth could not be evaluated in this study. The finding that iSP levels were in the same range regardless of the application of local anesthetic indicates that the quality or degree of this particular trauma did not induce release of significant amounts of this neurotransmitter. In the rat incisor pulp, CGRP seems to play an important role in vasodilatation because the specific blocker CGRP8-37 substantially reduced the duration of nerve-induced blood flow increase (14). With the use of the specific SP antagonist CP-96,345 nerveinduced vascular leakage in the pulp was reduced (17). As mentioned above, in immunohistochemical evaluation and in radioimmunoassay, different types of injuries induce distinct responses of CGRP and SP, suggesting a physiological role in the process of reactive vasodilatation and inflammatory response (5, 8, 9). The results of our study suggest that the neurovascular reaction of the human dental pulp is influenced by application of local anesthesia. This research received financial support from the dubil~iums-fund of the Austrian National Bank, no. 5062 and by the Austrian FWF, P09823M. Dr. Pert] is assistant professor, Department of Oral Surgery, School of Dental, Oral, and Maxillofacial Medicine, KarI-Franzens University of Graz, Austria and adjunct assistant professor, Department of Endodontics, School of Dental Medicine, University of Pennsylvania. Dr. Amann is associate professor, Department of Pharmacology, KarI-Franzens University of Graz. Dr. Odell is lecturer in Oral Medicine and Pathology, and Mr. Robinson is senior
lecturer Honorary Consultant, Department of Oral and Maxiilofacial Surgery, UMDS Guys Hospital, London. Dr. Kim is L. Grossman professor and chairman of the Department of Endodontics, School of Dental Medicine, University of Pennsylvania, Address requests for reprint requests to Christof Pertl, Universitaets-Zahnklinik, LKH Graz, A-8036 Graz, Austria.
References
1. Olgart L, Kerezoudis N. Nerve-pulp interactions. Arch Oral Biol 1994; 39:Suppl:47-54. 2. Olgart L, Edwall L, Gazelius B. Involvement of afferent nerves in pulpal blood-flow reaction in response to clinical and experimental procedures in the cat. Arch Oral Biol 1991;36:575-81. 3. N~,rhi M, Jyv&sj~rvi E, Virtanen A, Huopaniemi T, Ngassoa D, Hirvonen T. Role of intradental A- and C-type nerve fibers in dental pain mechanism. Proc Finn Dent Soc 1992;88 Suppl 1:507-16. 4. Holzer P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of Tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988;24:739-68. 5. Byers M, Taylor P, Khayat B, Kimberly C. Effects of injury and inflammation on pulpal and periapical nerves. J Endodon 1990;16:78-84. 6. Taylor P, Byers M, Redd P. Sprouting of CGRP nerve fibers in response to dentin injury in rat molars. Brain Res 1988;461:371-6. 7. Khayat B, Byers M. Responses of nerve fibers to pulpal inflammation and periapical lesions in rat molars demonstrated by calcitonin gene-related peptide immunocytochemistry. J Endodon 1988;14:577-87. 8. Grutzner E, Hargreaves K. Effect of injury on pulpal levels of immunereactive substance P and immunoreactive calcitonin gene-related peptide. J Endodon 1992;553-7. 9. Hargreaves K, Bowles W, Garry M. An in vitro method to evaluate regulation of neuropeptide release from dental pulp. J Endodon 1992;18:597600. 10. Pertl C, Liu M, Markowitz K, Kim S. Effects of capsaicin on KCI induced blood flow and sensory nerve activity changes in the tooth pulp. Pain 1993;52:351-58. 11. Heyeraas K, Kim S, Raab W, Byers M, Liu M. Effect of electrical tooth stimulation on blood flow, interstitial fluid pressure and substance P and CGRP-immunoreactive nerve fibers in the low compliant cat dental pulp. Microvasc Res 1994;47:329-43. 12, Silverman J, Kruger L. An interpretation of dental innervation based upon the pattern of calcitonin gene-related peptide(CGRP)-immunoreactive thin sensory axons. Somatosens Res 5:157-75. 13. Luthman J, Luthman D, H6kfelt T. Occurrence and distribution of different neurochemical markers in the human dental pulp. Arch Oral Biol 1992;37:193-208. 14. Kerezoudis N, Olgart L, Edwall L. CGRP(8-37) reduces the duration but not the maximal increase of antidromic vasodilation in dental pulp and lip of the rat. Acta Physiol Scand 1994;151:73-81. 15. Markowitz K, Bilotto G, Liu M, Jou Y, Kim S. Physiological studies of neurogenic inflammation in the dental pulp. J Endodon 1989;15:170. 16. Jebeles J, Reilly J, Gutierrez J, Bradley E, Kissin I. The effect of preincisional infiltration of tonsils with bupivacaine on the pain following tonsillectomy under general anesthesia. Pain 1991;47:305-08. 17. Kerezoudis N, Olgart L, Edwall L Involvement of substance P but not nitric oxide or calcitonin gene-related peptide in neurogenic plasma extravasation in rat incisor pulp and lip. Arch Oral Biol 1994;39:769-74.
Printed in U.S.A. VOL. 23, NO. 7, JULY 1997
Effects of Local Anesthesia on Substance P and CGRP Content of the Human Dental Pulp C. Pertl, R. Amann, E. Odell, P. D. Robinson, and S. Kim
The aim of the study was to determine immunoreactive Substance P (iSP) and immunoreactive calcitonin gene-related Peptide (iCGRP) content in the human dental pulp and whether local anesthesia has an effect on the neuropeptide content. Dental pulps were obtained from patients, who underwent surgical extraction of all 4 impacted wisdom teeth under general isoflurane gas anesthesia. There was a very high interindividual variation in tissue content, with small variation in levels found in teeth from the same patient. Pulps obtained from lower teeth without local anesthesia contained an average of 131 +_ 62 fmol/mg protein of iCGRP and 15 --- 9 fmol/mg iSP (n = 10). With additional mandibular block anesthesia the values were 194 _ 71 fmol/mg iCGRP (statistically significant, p = 0.0356, Mann-Whitney-Rank-Sum-Test) and 12 _+ 3.6 fmol/mg iSP. The results suggest that local anesthesia attenuates neuropeptide release in the human dental pulp during surgical extraction.
iCGRP using radioimmunoassays. Both pulpal exposure and acid etch produced significant decreases in tissue levels of iSP and iCGRP. Three to 10 days after injury iSP levels decreased to about 10% of baseline values, while iCGRP levels decreased to about 45% of baseline measures. With the method of in vitro superfusion of bovine dental pulp, Hargreaves et al. (9) found a 17-fold greater concentration of iCGRP than iSP. Administration of potassium chloride (50 mM) increased levels of iCGRP 3- to 5 times in the superfusate. It has further been shown that potassium-chloride-induced increase in pulpal blood flow was significantly inhibited following blockade of afferent neurons with capsaicin (10). The loss of iCGRP and iSP from electrically stimulated feline teeth pretreated with the sympathetic blocker guanethidine also suggests that these mediators may be responsible for vasodilatation (ll). The human dental pulp is richly innervated by both myelinated and unmyelinated afferent nerves. The subgroup of unmyelinated sensory fibers that contain CGRP and SP forms numerous endings in the coronal pulp and dentin and along many pulpal blood vessels (12, 13). Most of the studies in this field have been carried out in animals and their relevance to humans is unclear. The aims of this study were to determine SP and CGRP content in the human pulp after extraction under general anesthesia and to determine whether additional local anesthesia has an effect on their neuropeptide content.
Because the dental pulp resides in a rigid environment there is a need to control vasodilatation and to limit changes in tissue pressure that might disturb adequate blood perfusion. Changes in microcirculation and haemodynamics are naturally closely related to inflammatory responses. Various stimuli induce vasodilatation and extravasation and consequently increase interstitial pressure in the pulp of animals. It is very likely that these responses are mediated by release of neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) from fine caliber, capsaicin sensitive nerves (1, 2, 3, 4). Several studies by Byers and her colleagues (5, 6, 7) have evaluated the effects of different types of injuries on the immunohistochemical staining of peripheral CGRP fibers in the rat dental pulp. They found distinct neuropeptide responses, mild injuries producing an increase in sprouting of immunoreactive CGRP (iCGRP). Severe pulpal trauma resulted in either a reduction or an elimination of iCGRP staining. Grutzner et al. (8) investigated the effect of injury-induced changes in rat pulpal levels of both immunoreactive SP (iSP) and
METHODS Human dental pulps were obtained from patients who underwent surgical extraction of all 4 wisdom teeth under general isoflurane gas anesthesia. Only those teeth for which the following criteria were fulfilled were included: 1: similar (upright) position of left and right wisdom tooth; 2: full impaction; 3: similar length of operation on left and right tooth (-+5 min); 4: entire duration of the surgical extraction of one tooth (incision-extraction) did not exceed 15 min; and 5: teeth were completely intact after extraction. Local anesthetic with 1 carpule (1.8 ml) of prilocaine 4% without vasoconstrictor was given on the experimental side: buccal and lingual infiltration for the maxillary teeth and mandibular block for the mandibular teeth. The surgical procedure was performed in a conventional manner. Round burs at high speed were used for bone removal and
416
Local Anesthesia and Pulpal NeuropepUdes
Vol. 23, No. 7, July 1997 elevators for tooth extraction. All teeth were put in a perforated plastic tube and were frozen in liquid nitrogen instantly after extraction. During this procedure enamel and dentin fractured due to different thermal expansion of the different tooth tissues allowing easy removal of the coronal pulp in a frozen state. The coronal portions of 48 human wisdom tooth pulps obtained ti'om 14 patients were evaluated. Eight patients were female, six male, and their mean age was 17.7 (rain 15-max 22) years. The pulps were processed for radioimmunoassay and the results were calculated per mg of tissue protein content. For determination of iCGRP and iSP, samples were homogenized and extracted in 2 N acetic acid. An aliquot of the homogenate was used for protein determination (Bio-Rad). After centrifugation, the supernatants were lyophilized and used for radioimmunoassay, iCGRP was determined using antiserum RAS 6009N (Peninsula) and the radioligand (2-[125J]histididyl-10) CGRP (Amersham). iSP was determined using antiserum Rd2 and the radioligand [125J] BoltonHunter-substance P (Amersham). For high pressure liquid chromatography (HPLC), samples were pooled and injected on a reversed-phase nucleosil C18 column (Waters). Elution was performed by an 85-rain gradient from 20 to 60% acetonitrile at a flow rate of 1.5 ml/min. The fractions (1 rain) were lyophilized and assayed for CGRP as described above. Data are shown as means ± S.E.M.; statistical significance was determined using the Mann-Whitney Rank Sum Test (Sigma Stat statistical software, Jandel Scientific, Erkrath, Germany). Informed consent was obtained from all patients, and experiments were approved by the ethics committee of UMDS Guys and St. Thomas's Hospital, London.
RESULTS There was a large interindividual variation of tissue concentrations of iCGRP (10-850 fmol/mg protein) and iSP (1.17-127 fmol/mg protein) found in the control teeth, which had not been anesthetized with prilocaine. The neuropeptide content of different pulps in the same patient was much more constant. The difference in iCGRP or iSP content between different specimens from the same individual did not exceed a factor of 2.3. This was found with the exception of one patient where iCGRP of the upper third molar pulp was 5.2-fold higher than that of the corresponding mandibular. Pulps obtained from lower teeth without local anesthesia contained an average of 131 ± 62 fmol/mg iCGRP and 15 + 9 fmol/mg iSP (n = 10). With mandibular block anesthesia using one carpule prilocaine (4%) the values were 194 ± 71 fmol/mg iCGRP and 12 + 3.6 fmol/mg iSP. In the case of iCGRP the difference between control side and local anesthesia reached statistical significance (p = 0.0356, Mann Whitney Rank Sum Test). The identity of iCGRP was verified by HPLC: more than 90% of immunoreactivity eluted at the position of synthetic human-CGRP. When pulpal neuropeptide content of one side was compared with the contralateral tooth in the same patient, local anesthesia caused a considerable increase (Fig. 1). The neuropeptide content o]" pulps from upper control teeth contained 143 + 59 fmol/mg iCGRP and 9 -+ 3.7 fmol/mg iSP. Corresponding tissue levels of pulps on which side an additional buccal and lingual infiltration of a total of one carpule prilocaine 4% was given were 238 +_ 81 fmol/mg iCGRP and 18 _+ 10 fmol/mg iSP. Although there seemed to be a tendency for increased neuropeptide content after extraction under local anes-
417
300 t~ >
0) 200 +.J cO 0 0
100 Nerve block
Infiltration
FIG. 1. Pulpal neuropeptide content of the side with additional local anesthesia was compared with the contralateral tooth of the same
patient. Mandibular block anesthesia resulted in a iCGRP content of 227% (-+42%) and iSP level of 139% (4-16%) of the control side. The iCGRP and iSP levels under buccal and lingual infiltration were 153% (±20%) and 151% (+-18%) respectively. Statistical significance was calculated using raw data.
thetic infiltration (Fig. l), there was no statistically significant difference.
DISCUSSION In immunohistochemical studies of the human dental pulp more CGRP than SP fibers have been found. Both neuropeptides were found predominantly around pulpal blood vessels but also in the subodontoblastic region (l l, 12). Radioimmunoassay of tissue extracts from bovine dental pulp demonstrated a 17 times higher iCGRP level than iSP (9). We found a similar relationship in the evaluation of human coronal pulp tissue amounting to an approximately 10-fold higher concentration of CGRP than SP. We used a homogeneous group of extracted teeth including only completely intact, impacted third molars from 15 to 22 (mean 17.4) year old patients. Interestingly there was a very high interindividual variation in neuropeptide content. The stability of each individual's specific neuropeptide content is shown by the small variation in levels found in teeth from the same patient. It remains to be determined whether these findings have any physiological relevance such as individual dimension and different capacity of neurovascular mechanisms or different pain thresholds. There seemed to be no correlation in neuropeptide levels between roots with wide open apical foramina and those with more or less formed apices. This observation is in accordance with the immunohistochemical evaluation of Luthman et al. (12), who could not demonstrate any difference in the occurrence and distribution of iCGRP, iSP, and other neurochemical markers in pulps obtained from immature and fully formed teeth. The present study has also demonstrated that dynamic changes in neuropeptide content in the dental pulp occur in humans. The neurovascular reactions induced by noxious stimulation have been studied in numerous animal models. Electrical stimulation, CGRPand SP-infusion, application of capsaicin (the pungent ingredient of red peppers acts as a selective activator of unmyelinated afferent nerves), cavity preparation and percussion cause pulpal vasodilatation and an inflammatory response. Further, it has been shown that the effect of this variety of stimuli depend to a large extent on activation of afferent nerves (1, 3, 4). Application of local anes-
418
Journal of Endodontics
Pertl, et al.
thetic has been shown to block feline pulpal blood-flow increase induced by ultrasonic stimulation and KCI application to a deep dentinal cavity (2, 15). To study the effect of local anesthesia on neuropeptide release during the surgical removal of impacted wisdom teeth, mandibular block anesthesia and maxillary buccal and lingual infiltration were administered on one side. Local anesthesia given to patients in addition to general anesthesia is routinely used. It has been shown that this additional blockade of nociceptive input can decrease pain beyond the immediate postoperative period (16). We found a statistically significantly higher content of iCGRP under mandibular block anesthesia, suggesting that during the trauma of surgical extraction a smaller amount of the neurotransmitter was released, inactivated, and carried off by pulpal blood vessels than in corresponding control teeth. The same tendency was observed with infiltration of prilocaine around upper impacted wisdom teeth but did not reach statistical significance. Whether this is due to the different mechanism or amount of inhibition induced by either local infiltration or nerve block anesthesia or the greater surgical trauma required for extraction of lower impacted wisdom teeth could not be evaluated in this study. The finding that iSP levels were in the same range regardless of the application of local anesthetic indicates that the quality or degree of this particular trauma did not induce release of significant amounts of this neurotransmitter. In the rat incisor pulp, CGRP seems to play an important role in vasodilatation because the specific blocker CGRP8-37 substantially reduced the duration of nerve-induced blood flow increase (14). With the use of the specific SP antagonist CP-96,345 nerveinduced vascular leakage in the pulp was reduced (17). As mentioned above, in immunohistochemical evaluation and in radioimmunoassay, different types of injuries induce distinct responses of CGRP and SP, suggesting a physiological role in the process of reactive vasodilatation and inflammatory response (5, 8, 9). The results of our study suggest that the neurovascular reaction of the human dental pulp is influenced by application of local anesthesia. This research received financial support from the dubil~iums-fund of the Austrian National Bank, no. 5062 and by the Austrian FWF, P09823M. Dr. Pert] is assistant professor, Department of Oral Surgery, School of Dental, Oral, and Maxillofacial Medicine, KarI-Franzens University of Graz, Austria and adjunct assistant professor, Department of Endodontics, School of Dental Medicine, University of Pennsylvania. Dr. Amann is associate professor, Department of Pharmacology, KarI-Franzens University of Graz. Dr. Odell is lecturer in Oral Medicine and Pathology, and Mr. Robinson is senior
lecturer Honorary Consultant, Department of Oral and Maxiilofacial Surgery, UMDS Guys Hospital, London. Dr. Kim is L. Grossman professor and chairman of the Department of Endodontics, School of Dental Medicine, University of Pennsylvania, Address requests for reprint requests to Christof Pertl, Universitaets-Zahnklinik, LKH Graz, A-8036 Graz, Austria.
References
1. Olgart L, Kerezoudis N. Nerve-pulp interactions. Arch Oral Biol 1994; 39:Suppl:47-54. 2. Olgart L, Edwall L, Gazelius B. Involvement of afferent nerves in pulpal blood-flow reaction in response to clinical and experimental procedures in the cat. Arch Oral Biol 1991;36:575-81. 3. N~,rhi M, Jyv&sj~rvi E, Virtanen A, Huopaniemi T, Ngassoa D, Hirvonen T. Role of intradental A- and C-type nerve fibers in dental pain mechanism. Proc Finn Dent Soc 1992;88 Suppl 1:507-16. 4. Holzer P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of Tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988;24:739-68. 5. Byers M, Taylor P, Khayat B, Kimberly C. Effects of injury and inflammation on pulpal and periapical nerves. J Endodon 1990;16:78-84. 6. Taylor P, Byers M, Redd P. Sprouting of CGRP nerve fibers in response to dentin injury in rat molars. Brain Res 1988;461:371-6. 7. Khayat B, Byers M. Responses of nerve fibers to pulpal inflammation and periapical lesions in rat molars demonstrated by calcitonin gene-related peptide immunocytochemistry. J Endodon 1988;14:577-87. 8. Grutzner E, Hargreaves K. Effect of injury on pulpal levels of immunereactive substance P and immunoreactive calcitonin gene-related peptide. J Endodon 1992;553-7. 9. Hargreaves K, Bowles W, Garry M. An in vitro method to evaluate regulation of neuropeptide release from dental pulp. J Endodon 1992;18:597600. 10. Pertl C, Liu M, Markowitz K, Kim S. Effects of capsaicin on KCI induced blood flow and sensory nerve activity changes in the tooth pulp. Pain 1993;52:351-58. 11. Heyeraas K, Kim S, Raab W, Byers M, Liu M. Effect of electrical tooth stimulation on blood flow, interstitial fluid pressure and substance P and CGRP-immunoreactive nerve fibers in the low compliant cat dental pulp. Microvasc Res 1994;47:329-43. 12, Silverman J, Kruger L. An interpretation of dental innervation based upon the pattern of calcitonin gene-related peptide(CGRP)-immunoreactive thin sensory axons. Somatosens Res 5:157-75. 13. Luthman J, Luthman D, H6kfelt T. Occurrence and distribution of different neurochemical markers in the human dental pulp. Arch Oral Biol 1992;37:193-208. 14. Kerezoudis N, Olgart L, Edwall L. CGRP(8-37) reduces the duration but not the maximal increase of antidromic vasodilation in dental pulp and lip of the rat. Acta Physiol Scand 1994;151:73-81. 15. Markowitz K, Bilotto G, Liu M, Jou Y, Kim S. Physiological studies of neurogenic inflammation in the dental pulp. J Endodon 1989;15:170. 16. Jebeles J, Reilly J, Gutierrez J, Bradley E, Kissin I. The effect of preincisional infiltration of tonsils with bupivacaine on the pain following tonsillectomy under general anesthesia. Pain 1991;47:305-08. 17. Kerezoudis N, Olgart L, Edwall L Involvement of substance P but not nitric oxide or calcitonin gene-related peptide in neurogenic plasma extravasation in rat incisor pulp and lip. Arch Oral Biol 1994;39:769-74.