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Effect of injury on pulpal levels of immunoreactive substance P and immunoreactive calcitonin gene-related peptide
0099-2399/92/1811-0553/$03.00/0 JOURNAL OF ENDODONTICS Copyright © 1992 by The American Association of Endodontists
Printed in U.S.A.
VOL. 18, NO. 11, NOVEMBER1992
Effect of Injury on Pulpal Levels of Immunoreactive Substance P and Immunoreactive Calcitonin Gene-Related Peptide Eric H. Grutzner, DDS, MS, Mary G. Garry, PhD, and Kenneth M. Hargreaves, DDS, PhD
Neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P are present in dental pulp in relatively high concentrations. Previous studies have demonstrated that the staining density of immunoreactive CGRP (iCGRP) changes in dental pulp after tissue injury. This study evaluated injuryrelated changes in levels of both immunoreactive CGRP (iCGRP) and immunoreactive substance P (iSP) in dental pulp using radioimmunoassays. After pulpal exposure, iSP levels decreased to about 10% of baseline values, while iCGRP levels decreased to about 45% of baseline measures. After dentin exposure with acid etch, iSP levels decreased to about 10 to 20% of baseline measures, while iCGRP levels decreased to 60% of baseline values. For both forms of injury, iSP decreased to a greater extent than did iCGRP levels. Collectively, these findings indicate that pulpal neuropeptides undergo dynamic, injuryspecific, and peptide-specific responses following trauma to dental pulp.
Several studies by Byers and her colleagues (11-14) have evaluated the effects of different types of mechanical and infective injuries on the immunohistochemical staining of peripheral CGRP fibers in dental pulp. Mild injury to the pulp (e.g. cervical dentinal cavities) produced an increase in sprouting of iCGRP fibers after 4 days. Intermediate injuries, such as cavity preparation with smear layer removal, produced microabscess formation and sprouting of the iCGRP fibers near the area of injury. Severe injuries (e.g. pulpal exposure) resulted in either a reduction or elimination of iCGRP staining in 80% of examined teeth. These studies suggest that pulpal levels ofiCGRP display dynamic responses to injury. Since immunohistochemistry provides more precise information on peptide location rather than on concentration, we have evaluated neuropeptide responses to pulpal injury using the quantitative method of radioimmunoassay. In addition, we evaluated the responses of both iCGRP and iSP to two forms of dental injury, pulpal exposure and dentin exposure with acid etch. MATERIALS AND METHODS Male Sprague-Dawley rats (250 to 275 g) were housed (lights on: 0600 to 1800 h) for 7 days prior to the experiment with food and water available ad libitum. After an overnight fast, all surgeries were performed (1200 to 1700 h) using halothane anesthesia. Occlusal class I preparations were made using a modified #7002 bur in the three maxillary and three mandibular molars on one side. The bur was modified to ensure a constant depth of the preparation by welding an outer cylindrical bur guard (t 0-gauge stainless steel tubing) to permit either a 1.0-ram or 0.75-mm exposure of the bur. Preliminary studies indicated that a 1.0-mm preparation depth consistently resulted in a pulpal exposure while the 0.75-mm depth produced only a dentinal exposure. The dentinal exposure group was further treated with an acid etch using 40% H2PO4 (Ultra etch; Ultradent Products Inc., New York, NY) which was left in the preparation for 1 min. Rats generally recovered within 1 to 2 rain after surgery. Gross changes in eating and grooming habits were not observed in the postsurgical period. Rats were randomly divided into three groups as follows:
Neuropeptides such as immunoreactive substance P (iSP) and immunoreactive calcitonin gene-related peptide (iCGRP) are present in most oral tissues. In dental pulp, both iSP and iCGRP most likely originate from nociceptive primary afferent fibers, since their levels decrease after administration of capsaicin, a neurotoxin that can destroy a major subclass of nociceptors (primarily C polymodal and some A0 nociceptors) or after transection of the inferior alveolar nerve (1, 2). Pulpal neuropeptides are released from their nerve terminals after stimulation (3, 4) and may engage a number of targets to initiate or modulate the inflammatory response. Administration of substance P (SP) induces a biphasic response in pulpal blood flow (5), produces plasma extravasation (6), and evokes cytokine release from monocytes (7). Additional studies indicate that calcitonin gene-related peptide (CGRP) potentiates many of the effects of SP (8) and has led to the proposal that these neuropeptides contribute to the neurogenic component of inflammation (9, 10).
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group 1, untreated controls (n = 8), rats were anesthetized with halothane for 2 rain and two rats were killed at 3, 5, 7, or 10 days following the initial halothane anesthesia; group 2, pulp exposures were performed on separate groups of animals who were killed at either 3, 5, 7, or 10 days after exposure (n = 6/day); and group 3, dentinal exposure with acid etch were performed on separate groups of animals killed at 3, 5, 7, or 10 days after the preparation (n = 6/day). Tissue samples were collected after the rats were killed by CO2 asphyxiation. Adjacent first, second, and third molars were removed and stripped of all periodontal ligaments. The three molars from each quadrant were pooled. All samples were immediately placed in a 500-ul solution of 2 N acetic acid, 0.04 N HCI, and 2% ¢~-mercaptoethanol, boiled for 10 rain, homogenized (Teckmar Tissuemizer SDT 1810), and centrifuged. Aliquots of the supernatants were collected for subsequent radioimmunoassay (RIA). Residual supernatants were pooled and used for parallel dilution displacement curves and determination of sample nonspecific binding in the RIAs. All samples were frozen, lyophylized, and stored until assayed. The substance P and CGRP RIAs were previously validated. Samples were resuspended with 300 #1 of RIA buffer (0.15 M sodium phosphate, 0.05% bovine serum albumin, 0.02% sodium azide, 50 mg/100 ml bacitracin), vortexed, and assayed in duplicate. The SP antiserum (SP-1; a courtesy of Dr. S. Leeman) was used at a final dilution of 1:200,000. The CGRP antiserum (MI-l; kindly provided by Dr. M. Iadarola) was used at a final dilution of 1:1,000,000. Samples were preincubated for 48 h, then 100 ~tl of [125I-TyrS]substance P or [~25I-Tyr°]CGRP28_37 (approximately 20,000 to 25,000 cpm) were added to the solution, vortexed, and allowed to incubate for an additional 24 h. The RIA was stopped by the addition of 1 ml of a charcoal suspension (1.0:0.1 mix of activated charcoal:bovine serum albumin in a 0.55 % solution of RIA buffer) and centrifugation (25 min at 2000 rpm). The SP antiserum (SP-I) binds near the C-terminal sequence of the peptide. It shows no cross-reactivity (at 100 pM) with substance K, physalaemin, met-enkephalin, or eledoisin. The CGRP antiserum (MI-2) binds near the C-terminal end of CGRP, but does not cross-react with cholecystokinin, neuropeptide Y, or other peptides with similar C-terminal residues such as the molluscan cardioexcitatory (FMRFamide) peptides. Under nonequilibrium conditions, the minimum detection limit for all of these assays is approximately 1 fmol/tube, with 50% displacements of 4 to 6 fmol/tube. The inter- and intraassay coefficients of variation are less than 6 and 12%, respectively. Our preliminary studies indicated no significant difference between peptide levels in maxillary molars versus mandibular molars; in general, peptide levels did not vary by more than 10%. Accordingly, maxillary and mandibular data were averaged for each animal to determine individual mean femtomoles per tooth data prior to calculation of group mean values and statistical analyses. Statistical analysis of the group data consisted ol~using a two-way analysis of variance to determine the effects of time and injury for each of the dependent measures. A post hoc Duncan's multiple range test was used to determine whether injuries produced differences from control levels. Data are expressed as mean + SE. A difference was accepted as significant if the probability that it occurred due to chance was less than 5% (i.e. p < 0.05).
RESULTS Pulpal exposure resulted in a prolonged suppression in tissue levels of iSP (Fig. 1). By three days after exposure, levels of iSP decreased from baseline levels of 73.6 _.+ 10.3 fmol/ tooth to less than 10 fmol/tooth; this reduction in pulpal levels of iSP persisted throughout the 10-day observation period. The effect of pulpal exposure on levels of iSP was significant as indicated by analysis of variance (ANOVA) (F4,25 44.45; p < 0.0001). Pulpal exposure also resulted in a significant reduction in tissue levels of iCGRP (Fig. 2). Pulpal levels of iCGRP decreased from baseline values of 199.4 __+ 17.8 fmol/tooth to day 3 values of 86.1 + 6.4 fmol/tooth; this reduction was evident throughout the observation period. The effect of pulpal exposure on tissue levels of iCGRP was significant (ANOVA: F4.25 = 10.36; p < 0.0001). To permit a direct comparison of iSP responses to iCGRP responses following tissue injury, neuropeptide levels were normalized by dividing postinjury values by baseline measures (Table 1). Thus, the data for remaining pulpal content of neuropeptides are converted to percentage of baseline. These calculations indicate that iSP exhibited a significant decrease at all time points, as compared with changes in iCGRP (ANOVA: F~.2o= 316.6; p < 0.001) as compared with baseline values. Acid etch following dentin exposure also produced a significant reduction in pulpal levels of iSP (Fig. 3). Tissue levels ofiSP were reduced to nearly 10% of baseline levels by 3 days after acid etch. This reduction was maintained throughout the experimental period. The effect of acid etch on levels of iSP following acid etch was highly significant as indicated by (ANOVA: F4.2s = 35.52; p < 0.0001). The effect of dentin exposure with acid etch on tissue levels of iCGRP was also assessed (Fig. 4). Tissue levels of iCGRP =
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Time (days) FiG 1. Effect of pulpal exposure on levels of immunoreactive substance P in dental pulp. Rats (n = 6 to 8/group) were killed at 3 to 10 days after injury, and maxillary and mandibular teeth on the injured side were extracted with pulpal homogenates assayed for iSP by RIA. Error bars are SEM. **p < 0.01 as compared with baseline values.
Vol. 18, No. 11, November 1992
Pulpal Injury and Neuropeptides
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Time (days) FIG 2. Effect of pulpal exposure on levels of immunoreactive CGRP in dental pulp. Samples were collected from the same animals as described in the legend to Fig. 1. Error bars are SEM. ** p < 0.001 as compared with baseline values.
TABLE 1. Comparison of responses of iSP and iCGRP following pulpal exposure*
FIG 3. Effect of dentin exposure with acid etch on levels of immunoreactive substance P in dental pulp. Rats (n = 6 to 8/group) were killed at 3 to 10 days after acid etch, and maxillary and mandibular teeth on the injured side were extracted with pulpal homogenates assayed for iSP by RIA. Error bars are SEM. ** p < 0.01 as compared with baseline values.
Day Peptide
0 (%)
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iSP 100 1 2 . 0 + 0 . 1 5.8_+0.0 12.6+-0.0 11.7--.0.0 iCGRP 100 43.1 _+ 0.01" 44.0 _+ 0.21" 53.0 _+ 0.11" 52.0 +_ 0.01• Data are taken from Figs. 1 and 2 and are normalized to permit direct comparison between iSP and iCGRP (percentage of baseline was calculated by dividing pestinjury values by baseline values). 1 P < 0.01 versus SP.
were reduced to about 61% of baseline values by 3 days after acid etch, and this reduction was maintained through the 10day observation period. The effect of acid etch on pulpal levels of iCGRP was a significant finding (ANOVA: F4,25 = 10.26; p < 0.0001). Normalization of neuropeptide levels to baseline values permitted a direct comparison between remaining pulpal iSP and iCGRP after acid etch treatment (Table 2). The ANOVA revealed remaining iCGRP levels to be significantly greater than residual iSP values at all time points (FL]9 = 394.3; p < 0.001). A direct comparison of the effects of pulpal exposure to acid etch was also performed (compare Figs. 1 and 3). A twoway analysis of variance comparing these interventions for time-related effects indicate a borderline time-injury interaction (ANOVA: F~.39 = 2.89; p < 0.05). The post-hoc analysis by Duncan's multiple range test indicated that a difference between injuries was evident at the 10-day time point. A comparison was also made between the effects of exposure and acid etch on pulpal levels of iCGRP (compare Figs. 2 and 4). A two-way analysis of variance for time and injury indicates that a simple main effect occurred ( F k 3 9 = 7.12; p < 0.01). This simple main effect indicates that at all observed
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time points, pulpal levels of iCGRP were significantly lower following dental exposure as compared with acid etch. DISCUSSION This study evaluated the effects of pulpal injury on tissue levels of the neuropeptides, iSP and iCGRP. The results indicate that pulpal neuropeptides display distinct responses
556
Grutzner et al.
Journal of Endodontics TABLE 2. Comparison of responses of iSP and iCGRP following acid etch*
Day Peptide iSP iCGRP
0 C%) 100 100
3 (%) 9.7 _ 0.0 61.5 _+ 0.11"
5 (%) 11.6 + 0.1 55.8 _ 0.01"
7 (%) 14.4 +_ 0.0 61.2 + 0.01"
10 (%) 20.0 _+ 0.0 67.2 _ 0.01"
Data are taken from Figs. 3 and 4 and are normalized to permit direct comparisons between iSP and iCGRP (percentageof baselinewas calculatedby dividing postinjury values by baseline values). 1 P < 0,001 versus SP, •
to two types of tissue injury and that these responses vary depending on the neuropeptide examined. Both pulpal exposure and acid etch produced significant decreases in tissue levels of iSP and iCGRP. After pulpal exposure, iSP levels decreased to about 10% of baseline values, while iCGRP levels decreased to about 45% of baseline measures. After dentin exposure with acid etch, iSP levels decreased to about 10 to 20% of baseline measures, while iCGRP levels decreased to 60% of baseline values. For both forms of injury, iSP decreased to a greater extent than did iCGRP levels. Pulpal exposure produced a greater decrease in iCGRP levels over the entire 10-day observation period than acid etch. In contrast, a slight, but significant difference in iSP levels was evident only at the 10-day time point. Collectively, these findings indicate that pulpal neuropeptides undergo dynamic, injury-specific and peptide-specific responses following trauma to dental pulp. The study presented here was initiated following the elegant work of Byers and her colleagues (11-14), who have demonstrated that staining for iCGRP varies in response to pulpal injury. In general, their studies indicate that a substantial increase in CGRP staining occurs in regions adjacent to a mild inflammatory response. In contrast, there is a marked reduction in pulpal iCGRP staining after more severe forms of tissue injury (12, 14). For example, severe forms of pulpal injury results in either a reduction or elimination of iCGRP staining by 4 days after injury in 80% of examined teeth (14). The current study extends this work by evaluating iSP levels in addition to measuring iCGRP and by using a quantitative method (RIA) for determining neuropeptide levels. Both pulpal exposure and acid etch produced a substantial and prolonged reduction in tissue levels of iSP and iCGRP. The finding of a reduction in tissue content of these neuropeptides following injury may be due to several mechanisms. A reduction in tissue content of a neuropeptide may be due to increased release of the peptide from stored vesicles located in nerve terminal endings. For example, electrical stimulation of the inferior alveolar nerve releases iSP (3) and produces a corresponding reduction in pulpal content ofimmunoreactive neuropeptides (4). An alternative explanation for reduced tissue content of neuropeptides is an inhibition in either the synthesis of the neuropeptides at the cell bodies (located in the trigeminal ganglion) or an inhibition in the transport of neuropeptides along the nerve fiber. The present studies do not distinguish among these possible mechanisms. The second major finding in the present study was the differential response of iSP as compared with iCGRP following pulpal injuries. This difference is not attributable to interanimal variation since both peptides were measured from aliquots taken from the same teeth. Rather, this difference
must be due to a difference in the biology of these neuropeptides. One interpretation consistent with these findings is a differential distribution of iSP- as compared with iCGRPcontaining fibers. Numerous studies examining sensory ganglia by the method of double-labeling immunohistochemistry have demonstrated that virtually all iSP-containing cell bodies also contain iCGRP. However, in addition, there is an appreciable population of iCGRP-containing fibers which do not contain iSP. In general, this latter population consists of large diameter (30- to 45-tzm) ganglion cell bodies (15). Additional studies have shown that peripheral trigeminal fibers exhibit this disparity in staining for iSP and iCGRP. For example, a subset of nerves in the rat iris consist ofa CGRP-positive, SPnegative fiber network which is resistant to the neurotoxin capsaicin (16). This suggests that iCGRP can reside in a subpopulation of peripheral trigeminal nerves which do not contain iSP. The observation that iCGRP exists in fibers with or without iSP has led to the hypothesis that peripheral neurogenic inflammation may be due, at least in part, to differential activation of various populations of peripheral nerve terminals. In this model, peripheral nerve terminals may be capable of releasing CGRP either with SP or with other neuromediators (9, 10). Based on the study presented here, one assumption of this hypothesis is that the neurons containing both iSP and iCGRP show a greater responsiveness to tissue injury than those containing iCGRP alone. Under these conditions, iSP would have a greater reduction in tissue content following pulpal injury as compared with iCGRP levels. However, additional hypotheses are possible and include a differential release of neuropeptides from nerve endings located in dental pulp or differences in biological half-life in dental pulp following secretion. Moreover, the trigeminal ganglion may display greater levels of iCGRP synthesis in response to tissue injury as compared with iSP, thereby maintaining greater pulpal content of iCGRP. The present study does not distinguish among these alternative hypotheses, but does indicate that dental pulp injury may prove to be a useful model for studies examining differences in neuropeptide biology in response to tissue injury. The final finding of the present study was the distinct neuropeptide responses to pulpal exposure as compared with acid etch. At all time points examined, pulpal exposure produced a substantially greater reduction in iCGRP levels as compared with acid etch. This difference between the two forms of injury was highly significant and persisted throughout the 10-day observation period. This is consistent with previous observations of Byers and her colleagues, who categorized pulpal injury as mild, intermediate, or severe, depending on the inflammatory response (11). In that system,
Vol. 18, No. 11, November 1992
acid etch produced an intermediate level of damage with sprouting ofiCGRP fibers near the abscess. In contrast, pulpal exposure was categorized as a severe form of tissue injury with necrosis leading to loss of iCGRP staining in the pulpal tissue. Although this is consistent with our iCGRP results, it is not clear why there is a lack of a corresponding difference in iSP levels between the two forms of tissue injury. This may be due to the statistical concept of the "floor" effect, in which it is difficult to distinguish between two interventions when both produce results near the minimal point of measurement. For example, it is possible that nearly all iSP fibers are destroyed with both forms of tissue injury and that residual pulpal content measured arises solely from a vascular origin
(17). Together, these results illustrate the complex flexibility of the peripheral nervous system. Neuropeptide responses are not invariant with different forms of tissue injury. Rather, neuropeptide responses depend on the type and possibly the magnitude of tissue injury. In this case, pulpal exposure produced a significantly greater depletion of tissue content of iCGRP as compared with dentin exposure with acid etch. These studies indicate that a significant change occurs in pulpal content of neuropeptides in response to tissue injury. The clinical implications of these findings are derived from the physiological properties of these neuropeptides. Peripheral administration of SP and CGRP produces a number of actions which are thought to modulate the development of inflammation and promote the healing response. Initial release of relatively high concentrations of these neuropeptides may contribute to the development of pulpitis through their vasoactive effects. For example, previous studies have demonstrated that administration of SP induces a biphasic blood flow response in pulp with an initial increase in pulpal blood flow followed by a sustained decrease in blood flow (5). Additional studies have demonstrated that co-administration of SP with CGRP produces a potentiation in these pulpal vascular responses (8). These actions have lent credence to the hypothesis that release of these neuropeptides in peripheral tissue leads to the development of a neurogenically mediated inflammation. However, additional studies have demonstrated that the release of these neuropeptides actually promotes the healing process. For example, studies conducted in both rats and pigs have demonstrated that administration of CGRP significantly increases the survival of both pedicle (i.e. attached) and island flaps elevated from the skin (I 8, 19). Conversely, pretreating rats with the neurotoxin capsaicin causes a significant depletion of both iCGRP and iSP and, at the same time, significantly reduces the survival of skin flaps in rats (20). These latter findings indicate that the release of neuropeptides into the injured tissue actually promotes the healing response and appears to be necessary for a normal healing response. Thus, the neuropeptides CGRP and SP appear to be capable of playing critical roles in mediating tissue responses to physical injury. These physiological properties suggest a wide avenue of clinical applications, since modulation of neuropeptide responses may well promote pulpal healing in response to injuries such as exposure or acid etch. One potential applica-
Pulpal Injury and Neuropeptides
557
tion for this type of therapy would be the inclusion of slowrelease neuroactive agents as a part of deep cavity liners, or as a component of direct or indirect pulp-capping medicaments. The modulation of neuropeptide responses to tissue injury may well provide a new approach for treating pulpal injury. This research was supported in part by NRSA fellowship F32-DE05606 (to M. G. G.). Dr. Grutzner and Dr. Garry are members of the Department of Restorative Sciences, Division of Endodontics, and Dr. Hargreaves is a member of the Department of Restorative Sciences, Division of Endodontics and Department of Pharmacology, University of Minnesota School of Dentistry, Minneapolis, MN. Address requests for reprints to Dr. Kenneth Hargreaves, School of Dentistry, Moos Health Sciences Tower, 515 Delaware St., SE, Minneapolis, MN 55455.
References 1. Olgart L, Hokfelt T, Nilsson G, Pernow B. Localization of substance Plike immunoreactivity in nerves in the tooth pulp. Pain 1977;4:153-9. 2. Wakisaka S, Nishikawa S, Ichikawa H, Matsuo S, Takano Y, Akai M. The distribution and origin of SP-like immunomactivity in rat molar pulp and periodontal tissues. Arch Oral Bio11985;30:813-15. 3. Brodin E, Nilsson G. Tissue concentration and release of substance Plike immunoreactivity in the dental pulp. Acta Physiol Scand 1981 ;111:141-9. 4. Gazelius B, Brodin E, Olgart L. Depletion of substance P-like immunereactivity in the oat dental pulp by antidromic nerve stimulation. Acta Physiol Scand 1981 ;111:419-27. 5. Kim S, Dorocher-Kim, Liu M-T, Trowbridge H. Biphasic pulp blood flow response to SP in the dog as measured with a radiolabled microsphere injection method. Arch Oral Bio11988;33:305-9. 6. Chahl L. Interactions of SP with putative mediators of inflammation and ATP. Eur J Pharmaco11977;44:45-9. 7. Lotz M, Vaughan J, Carson D. Effect of neuropeptides on production of inflammatory cytokines by human monocytes. Science 1988;241:1218-21. 8. Gazelius B, Edwall B, Olgart L, Lundberg M, Hokfelt T, Fischer J. Vasodilatory effects and coexistence of calcitonin gene-related peptide and substance P in sensory nerves of cat dental pulp. Acta Physiol Scand 1987; 130:33-40. 9. Kim S. Neurovascular interactions in the dental pulp in health and inflammation. J Endodon 1990; 16:48-53. 10. Saria A, Gamse R, Lundberg J, et el. Co-existence of tachykinins and calcitonin gene-related peptide in sensory nerves in relation to neurogenic inflammation. In: Haakinson R, Sundler F, eds. Tachykinin antagonists. Amsterdam: Elsevier Sciences Publications, 1985. 11. Byers M, Taylor P, Khayat B, Kimberly C. Effects of injury and inflammation on pulpal and periapical nerves. J Endodon 1990;16:78-84. 12. Khayat B, Byers M. Responses of nerve fibers to pulpal inflammation and periapical lesions in rat molars demonstrated by cacitonin gene-related peptide immunocytochemistry. J Endodon 1988;14:577-87. 13. Kimbedy C, Byers M. Inflammation of rat molar pulp and poriodontium causes increased calcitonin gene related peptide and axonal sprouting. Anat Rec 1988;222:289-300. 14. 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. 15. Ju G, Hokfelt T, Brodin E, et el. Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide-, and cholecystokinin-immunoreactive cells. Cell Tissue Res 1981 ;247:417-31. 16. Matsuyama T, Wanaka A, Yoneda S, et el. Two distinct calcitonin generelated poptide-containing peripheral nervous systems: distribution and quantitative difference between the iris and cerebral artery with specific reference to substance P. Brain Res 1986;373:205-12. 17. Zaidi M, Bevis P, Girgis S, Lynch C, Stevenso J, Maclntyre I. Circulating CGRP comes from the perivascular nerves. Eur J Pharmacol 1985;117: 283-4. 18. Heden P, Jernbeck J, Kjartansson J, Samuelson U. Increased skin flap survival and arterial dilation by calcitonin gene-related poptide. Scand J Plast Reconstr Surg 1989;23:11-6. 19. Kjartansson J, Dalsgaard C. Calcitonin gene-related peptide increases survival of a musculocutaneous critical flap in the rat. Eur J Pharmacol 1987; 142:355-8. 20. Kjartansson J, Dalsgaard C, Jonsson X. Decreased survival of experimental critical flaps in rats after sensory denervation with capsaicin. Plast Reconstr Surg 1987;79:218-221.
Printed in U.S.A.
VOL. 18, NO. 11, NOVEMBER1992
Effect of Injury on Pulpal Levels of Immunoreactive Substance P and Immunoreactive Calcitonin Gene-Related Peptide Eric H. Grutzner, DDS, MS, Mary G. Garry, PhD, and Kenneth M. Hargreaves, DDS, PhD
Neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P are present in dental pulp in relatively high concentrations. Previous studies have demonstrated that the staining density of immunoreactive CGRP (iCGRP) changes in dental pulp after tissue injury. This study evaluated injuryrelated changes in levels of both immunoreactive CGRP (iCGRP) and immunoreactive substance P (iSP) in dental pulp using radioimmunoassays. After pulpal exposure, iSP levels decreased to about 10% of baseline values, while iCGRP levels decreased to about 45% of baseline measures. After dentin exposure with acid etch, iSP levels decreased to about 10 to 20% of baseline measures, while iCGRP levels decreased to 60% of baseline values. For both forms of injury, iSP decreased to a greater extent than did iCGRP levels. Collectively, these findings indicate that pulpal neuropeptides undergo dynamic, injuryspecific, and peptide-specific responses following trauma to dental pulp.
Several studies by Byers and her colleagues (11-14) have evaluated the effects of different types of mechanical and infective injuries on the immunohistochemical staining of peripheral CGRP fibers in dental pulp. Mild injury to the pulp (e.g. cervical dentinal cavities) produced an increase in sprouting of iCGRP fibers after 4 days. Intermediate injuries, such as cavity preparation with smear layer removal, produced microabscess formation and sprouting of the iCGRP fibers near the area of injury. Severe injuries (e.g. pulpal exposure) resulted in either a reduction or elimination of iCGRP staining in 80% of examined teeth. These studies suggest that pulpal levels ofiCGRP display dynamic responses to injury. Since immunohistochemistry provides more precise information on peptide location rather than on concentration, we have evaluated neuropeptide responses to pulpal injury using the quantitative method of radioimmunoassay. In addition, we evaluated the responses of both iCGRP and iSP to two forms of dental injury, pulpal exposure and dentin exposure with acid etch. MATERIALS AND METHODS Male Sprague-Dawley rats (250 to 275 g) were housed (lights on: 0600 to 1800 h) for 7 days prior to the experiment with food and water available ad libitum. After an overnight fast, all surgeries were performed (1200 to 1700 h) using halothane anesthesia. Occlusal class I preparations were made using a modified #7002 bur in the three maxillary and three mandibular molars on one side. The bur was modified to ensure a constant depth of the preparation by welding an outer cylindrical bur guard (t 0-gauge stainless steel tubing) to permit either a 1.0-ram or 0.75-mm exposure of the bur. Preliminary studies indicated that a 1.0-mm preparation depth consistently resulted in a pulpal exposure while the 0.75-mm depth produced only a dentinal exposure. The dentinal exposure group was further treated with an acid etch using 40% H2PO4 (Ultra etch; Ultradent Products Inc., New York, NY) which was left in the preparation for 1 min. Rats generally recovered within 1 to 2 rain after surgery. Gross changes in eating and grooming habits were not observed in the postsurgical period. Rats were randomly divided into three groups as follows:
Neuropeptides such as immunoreactive substance P (iSP) and immunoreactive calcitonin gene-related peptide (iCGRP) are present in most oral tissues. In dental pulp, both iSP and iCGRP most likely originate from nociceptive primary afferent fibers, since their levels decrease after administration of capsaicin, a neurotoxin that can destroy a major subclass of nociceptors (primarily C polymodal and some A0 nociceptors) or after transection of the inferior alveolar nerve (1, 2). Pulpal neuropeptides are released from their nerve terminals after stimulation (3, 4) and may engage a number of targets to initiate or modulate the inflammatory response. Administration of substance P (SP) induces a biphasic response in pulpal blood flow (5), produces plasma extravasation (6), and evokes cytokine release from monocytes (7). Additional studies indicate that calcitonin gene-related peptide (CGRP) potentiates many of the effects of SP (8) and has led to the proposal that these neuropeptides contribute to the neurogenic component of inflammation (9, 10).
553
554
Journal of Endodontics
Grutzner et al.
group 1, untreated controls (n = 8), rats were anesthetized with halothane for 2 rain and two rats were killed at 3, 5, 7, or 10 days following the initial halothane anesthesia; group 2, pulp exposures were performed on separate groups of animals who were killed at either 3, 5, 7, or 10 days after exposure (n = 6/day); and group 3, dentinal exposure with acid etch were performed on separate groups of animals killed at 3, 5, 7, or 10 days after the preparation (n = 6/day). Tissue samples were collected after the rats were killed by CO2 asphyxiation. Adjacent first, second, and third molars were removed and stripped of all periodontal ligaments. The three molars from each quadrant were pooled. All samples were immediately placed in a 500-ul solution of 2 N acetic acid, 0.04 N HCI, and 2% ¢~-mercaptoethanol, boiled for 10 rain, homogenized (Teckmar Tissuemizer SDT 1810), and centrifuged. Aliquots of the supernatants were collected for subsequent radioimmunoassay (RIA). Residual supernatants were pooled and used for parallel dilution displacement curves and determination of sample nonspecific binding in the RIAs. All samples were frozen, lyophylized, and stored until assayed. The substance P and CGRP RIAs were previously validated. Samples were resuspended with 300 #1 of RIA buffer (0.15 M sodium phosphate, 0.05% bovine serum albumin, 0.02% sodium azide, 50 mg/100 ml bacitracin), vortexed, and assayed in duplicate. The SP antiserum (SP-1; a courtesy of Dr. S. Leeman) was used at a final dilution of 1:200,000. The CGRP antiserum (MI-l; kindly provided by Dr. M. Iadarola) was used at a final dilution of 1:1,000,000. Samples were preincubated for 48 h, then 100 ~tl of [125I-TyrS]substance P or [~25I-Tyr°]CGRP28_37 (approximately 20,000 to 25,000 cpm) were added to the solution, vortexed, and allowed to incubate for an additional 24 h. The RIA was stopped by the addition of 1 ml of a charcoal suspension (1.0:0.1 mix of activated charcoal:bovine serum albumin in a 0.55 % solution of RIA buffer) and centrifugation (25 min at 2000 rpm). The SP antiserum (SP-I) binds near the C-terminal sequence of the peptide. It shows no cross-reactivity (at 100 pM) with substance K, physalaemin, met-enkephalin, or eledoisin. The CGRP antiserum (MI-2) binds near the C-terminal end of CGRP, but does not cross-react with cholecystokinin, neuropeptide Y, or other peptides with similar C-terminal residues such as the molluscan cardioexcitatory (FMRFamide) peptides. Under nonequilibrium conditions, the minimum detection limit for all of these assays is approximately 1 fmol/tube, with 50% displacements of 4 to 6 fmol/tube. The inter- and intraassay coefficients of variation are less than 6 and 12%, respectively. Our preliminary studies indicated no significant difference between peptide levels in maxillary molars versus mandibular molars; in general, peptide levels did not vary by more than 10%. Accordingly, maxillary and mandibular data were averaged for each animal to determine individual mean femtomoles per tooth data prior to calculation of group mean values and statistical analyses. Statistical analysis of the group data consisted ol~using a two-way analysis of variance to determine the effects of time and injury for each of the dependent measures. A post hoc Duncan's multiple range test was used to determine whether injuries produced differences from control levels. Data are expressed as mean + SE. A difference was accepted as significant if the probability that it occurred due to chance was less than 5% (i.e. p < 0.05).
RESULTS Pulpal exposure resulted in a prolonged suppression in tissue levels of iSP (Fig. 1). By three days after exposure, levels of iSP decreased from baseline levels of 73.6 _.+ 10.3 fmol/ tooth to less than 10 fmol/tooth; this reduction in pulpal levels of iSP persisted throughout the 10-day observation period. The effect of pulpal exposure on levels of iSP was significant as indicated by analysis of variance (ANOVA) (F4,25 44.45; p < 0.0001). Pulpal exposure also resulted in a significant reduction in tissue levels of iCGRP (Fig. 2). Pulpal levels of iCGRP decreased from baseline values of 199.4 __+ 17.8 fmol/tooth to day 3 values of 86.1 + 6.4 fmol/tooth; this reduction was evident throughout the observation period. The effect of pulpal exposure on tissue levels of iCGRP was significant (ANOVA: F4.25 = 10.36; p < 0.0001). To permit a direct comparison of iSP responses to iCGRP responses following tissue injury, neuropeptide levels were normalized by dividing postinjury values by baseline measures (Table 1). Thus, the data for remaining pulpal content of neuropeptides are converted to percentage of baseline. These calculations indicate that iSP exhibited a significant decrease at all time points, as compared with changes in iCGRP (ANOVA: F~.2o= 316.6; p < 0.001) as compared with baseline values. Acid etch following dentin exposure also produced a significant reduction in pulpal levels of iSP (Fig. 3). Tissue levels ofiSP were reduced to nearly 10% of baseline levels by 3 days after acid etch. This reduction was maintained throughout the experimental period. The effect of acid etch on levels of iSP following acid etch was highly significant as indicated by (ANOVA: F4.2s = 35.52; p < 0.0001). The effect of dentin exposure with acid etch on tissue levels of iCGRP was also assessed (Fig. 4). Tissue levels of iCGRP =
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Vol. 18, No. 11, November 1992
Pulpal Injury and Neuropeptides
555
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TABLE 1. Comparison of responses of iSP and iCGRP following pulpal exposure*
FIG 3. Effect of dentin exposure with acid etch on levels of immunoreactive substance P in dental pulp. Rats (n = 6 to 8/group) were killed at 3 to 10 days after acid etch, and maxillary and mandibular teeth on the injured side were extracted with pulpal homogenates assayed for iSP by RIA. Error bars are SEM. ** p < 0.01 as compared with baseline values.
Day Peptide
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iSP 100 1 2 . 0 + 0 . 1 5.8_+0.0 12.6+-0.0 11.7--.0.0 iCGRP 100 43.1 _+ 0.01" 44.0 _+ 0.21" 53.0 _+ 0.11" 52.0 +_ 0.01• Data are taken from Figs. 1 and 2 and are normalized to permit direct comparison between iSP and iCGRP (percentage of baseline was calculated by dividing pestinjury values by baseline values). 1 P < 0.01 versus SP.
were reduced to about 61% of baseline values by 3 days after acid etch, and this reduction was maintained through the 10day observation period. The effect of acid etch on pulpal levels of iCGRP was a significant finding (ANOVA: F4,25 = 10.26; p < 0.0001). Normalization of neuropeptide levels to baseline values permitted a direct comparison between remaining pulpal iSP and iCGRP after acid etch treatment (Table 2). The ANOVA revealed remaining iCGRP levels to be significantly greater than residual iSP values at all time points (FL]9 = 394.3; p < 0.001). A direct comparison of the effects of pulpal exposure to acid etch was also performed (compare Figs. 1 and 3). A twoway analysis of variance comparing these interventions for time-related effects indicate a borderline time-injury interaction (ANOVA: F~.39 = 2.89; p < 0.05). The post-hoc analysis by Duncan's multiple range test indicated that a difference between injuries was evident at the 10-day time point. A comparison was also made between the effects of exposure and acid etch on pulpal levels of iCGRP (compare Figs. 2 and 4). A two-way analysis of variance for time and injury indicates that a simple main effect occurred ( F k 3 9 = 7.12; p < 0.01). This simple main effect indicates that at all observed
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time points, pulpal levels of iCGRP were significantly lower following dental exposure as compared with acid etch. DISCUSSION This study evaluated the effects of pulpal injury on tissue levels of the neuropeptides, iSP and iCGRP. The results indicate that pulpal neuropeptides display distinct responses
556
Grutzner et al.
Journal of Endodontics TABLE 2. Comparison of responses of iSP and iCGRP following acid etch*
Day Peptide iSP iCGRP
0 C%) 100 100
3 (%) 9.7 _ 0.0 61.5 _+ 0.11"
5 (%) 11.6 + 0.1 55.8 _ 0.01"
7 (%) 14.4 +_ 0.0 61.2 + 0.01"
10 (%) 20.0 _+ 0.0 67.2 _ 0.01"
Data are taken from Figs. 3 and 4 and are normalized to permit direct comparisons between iSP and iCGRP (percentageof baselinewas calculatedby dividing postinjury values by baseline values). 1 P < 0,001 versus SP, •
to two types of tissue injury and that these responses vary depending on the neuropeptide examined. Both pulpal exposure and acid etch produced significant decreases in tissue levels of iSP and iCGRP. After pulpal exposure, iSP levels decreased to about 10% of baseline values, while iCGRP levels decreased to about 45% of baseline measures. After dentin exposure with acid etch, iSP levels decreased to about 10 to 20% of baseline measures, while iCGRP levels decreased to 60% of baseline values. For both forms of injury, iSP decreased to a greater extent than did iCGRP levels. Pulpal exposure produced a greater decrease in iCGRP levels over the entire 10-day observation period than acid etch. In contrast, a slight, but significant difference in iSP levels was evident only at the 10-day time point. Collectively, these findings indicate that pulpal neuropeptides undergo dynamic, injury-specific and peptide-specific responses following trauma to dental pulp. The study presented here was initiated following the elegant work of Byers and her colleagues (11-14), who have demonstrated that staining for iCGRP varies in response to pulpal injury. In general, their studies indicate that a substantial increase in CGRP staining occurs in regions adjacent to a mild inflammatory response. In contrast, there is a marked reduction in pulpal iCGRP staining after more severe forms of tissue injury (12, 14). For example, severe forms of pulpal injury results in either a reduction or elimination of iCGRP staining by 4 days after injury in 80% of examined teeth (14). The current study extends this work by evaluating iSP levels in addition to measuring iCGRP and by using a quantitative method (RIA) for determining neuropeptide levels. Both pulpal exposure and acid etch produced a substantial and prolonged reduction in tissue levels of iSP and iCGRP. The finding of a reduction in tissue content of these neuropeptides following injury may be due to several mechanisms. A reduction in tissue content of a neuropeptide may be due to increased release of the peptide from stored vesicles located in nerve terminal endings. For example, electrical stimulation of the inferior alveolar nerve releases iSP (3) and produces a corresponding reduction in pulpal content ofimmunoreactive neuropeptides (4). An alternative explanation for reduced tissue content of neuropeptides is an inhibition in either the synthesis of the neuropeptides at the cell bodies (located in the trigeminal ganglion) or an inhibition in the transport of neuropeptides along the nerve fiber. The present studies do not distinguish among these possible mechanisms. The second major finding in the present study was the differential response of iSP as compared with iCGRP following pulpal injuries. This difference is not attributable to interanimal variation since both peptides were measured from aliquots taken from the same teeth. Rather, this difference
must be due to a difference in the biology of these neuropeptides. One interpretation consistent with these findings is a differential distribution of iSP- as compared with iCGRPcontaining fibers. Numerous studies examining sensory ganglia by the method of double-labeling immunohistochemistry have demonstrated that virtually all iSP-containing cell bodies also contain iCGRP. However, in addition, there is an appreciable population of iCGRP-containing fibers which do not contain iSP. In general, this latter population consists of large diameter (30- to 45-tzm) ganglion cell bodies (15). Additional studies have shown that peripheral trigeminal fibers exhibit this disparity in staining for iSP and iCGRP. For example, a subset of nerves in the rat iris consist ofa CGRP-positive, SPnegative fiber network which is resistant to the neurotoxin capsaicin (16). This suggests that iCGRP can reside in a subpopulation of peripheral trigeminal nerves which do not contain iSP. The observation that iCGRP exists in fibers with or without iSP has led to the hypothesis that peripheral neurogenic inflammation may be due, at least in part, to differential activation of various populations of peripheral nerve terminals. In this model, peripheral nerve terminals may be capable of releasing CGRP either with SP or with other neuromediators (9, 10). Based on the study presented here, one assumption of this hypothesis is that the neurons containing both iSP and iCGRP show a greater responsiveness to tissue injury than those containing iCGRP alone. Under these conditions, iSP would have a greater reduction in tissue content following pulpal injury as compared with iCGRP levels. However, additional hypotheses are possible and include a differential release of neuropeptides from nerve endings located in dental pulp or differences in biological half-life in dental pulp following secretion. Moreover, the trigeminal ganglion may display greater levels of iCGRP synthesis in response to tissue injury as compared with iSP, thereby maintaining greater pulpal content of iCGRP. The present study does not distinguish among these alternative hypotheses, but does indicate that dental pulp injury may prove to be a useful model for studies examining differences in neuropeptide biology in response to tissue injury. The final finding of the present study was the distinct neuropeptide responses to pulpal exposure as compared with acid etch. At all time points examined, pulpal exposure produced a substantially greater reduction in iCGRP levels as compared with acid etch. This difference between the two forms of injury was highly significant and persisted throughout the 10-day observation period. This is consistent with previous observations of Byers and her colleagues, who categorized pulpal injury as mild, intermediate, or severe, depending on the inflammatory response (11). In that system,
Vol. 18, No. 11, November 1992
acid etch produced an intermediate level of damage with sprouting ofiCGRP fibers near the abscess. In contrast, pulpal exposure was categorized as a severe form of tissue injury with necrosis leading to loss of iCGRP staining in the pulpal tissue. Although this is consistent with our iCGRP results, it is not clear why there is a lack of a corresponding difference in iSP levels between the two forms of tissue injury. This may be due to the statistical concept of the "floor" effect, in which it is difficult to distinguish between two interventions when both produce results near the minimal point of measurement. For example, it is possible that nearly all iSP fibers are destroyed with both forms of tissue injury and that residual pulpal content measured arises solely from a vascular origin
(17). Together, these results illustrate the complex flexibility of the peripheral nervous system. Neuropeptide responses are not invariant with different forms of tissue injury. Rather, neuropeptide responses depend on the type and possibly the magnitude of tissue injury. In this case, pulpal exposure produced a significantly greater depletion of tissue content of iCGRP as compared with dentin exposure with acid etch. These studies indicate that a significant change occurs in pulpal content of neuropeptides in response to tissue injury. The clinical implications of these findings are derived from the physiological properties of these neuropeptides. Peripheral administration of SP and CGRP produces a number of actions which are thought to modulate the development of inflammation and promote the healing response. Initial release of relatively high concentrations of these neuropeptides may contribute to the development of pulpitis through their vasoactive effects. For example, previous studies have demonstrated that administration of SP induces a biphasic blood flow response in pulp with an initial increase in pulpal blood flow followed by a sustained decrease in blood flow (5). Additional studies have demonstrated that co-administration of SP with CGRP produces a potentiation in these pulpal vascular responses (8). These actions have lent credence to the hypothesis that release of these neuropeptides in peripheral tissue leads to the development of a neurogenically mediated inflammation. However, additional studies have demonstrated that the release of these neuropeptides actually promotes the healing process. For example, studies conducted in both rats and pigs have demonstrated that administration of CGRP significantly increases the survival of both pedicle (i.e. attached) and island flaps elevated from the skin (I 8, 19). Conversely, pretreating rats with the neurotoxin capsaicin causes a significant depletion of both iCGRP and iSP and, at the same time, significantly reduces the survival of skin flaps in rats (20). These latter findings indicate that the release of neuropeptides into the injured tissue actually promotes the healing response and appears to be necessary for a normal healing response. Thus, the neuropeptides CGRP and SP appear to be capable of playing critical roles in mediating tissue responses to physical injury. These physiological properties suggest a wide avenue of clinical applications, since modulation of neuropeptide responses may well promote pulpal healing in response to injuries such as exposure or acid etch. One potential applica-
Pulpal Injury and Neuropeptides
557
tion for this type of therapy would be the inclusion of slowrelease neuroactive agents as a part of deep cavity liners, or as a component of direct or indirect pulp-capping medicaments. The modulation of neuropeptide responses to tissue injury may well provide a new approach for treating pulpal injury. This research was supported in part by NRSA fellowship F32-DE05606 (to M. G. G.). Dr. Grutzner and Dr. Garry are members of the Department of Restorative Sciences, Division of Endodontics, and Dr. Hargreaves is a member of the Department of Restorative Sciences, Division of Endodontics and Department of Pharmacology, University of Minnesota School of Dentistry, Minneapolis, MN. Address requests for reprints to Dr. Kenneth Hargreaves, School of Dentistry, Moos Health Sciences Tower, 515 Delaware St., SE, Minneapolis, MN 55455.
References 1. Olgart L, Hokfelt T, Nilsson G, Pernow B. Localization of substance Plike immunoreactivity in nerves in the tooth pulp. Pain 1977;4:153-9. 2. Wakisaka S, Nishikawa S, Ichikawa H, Matsuo S, Takano Y, Akai M. The distribution and origin of SP-like immunomactivity in rat molar pulp and periodontal tissues. Arch Oral Bio11985;30:813-15. 3. Brodin E, Nilsson G. Tissue concentration and release of substance Plike immunoreactivity in the dental pulp. Acta Physiol Scand 1981 ;111:141-9. 4. Gazelius B, Brodin E, Olgart L. Depletion of substance P-like immunereactivity in the oat dental pulp by antidromic nerve stimulation. Acta Physiol Scand 1981 ;111:419-27. 5. Kim S, Dorocher-Kim, Liu M-T, Trowbridge H. Biphasic pulp blood flow response to SP in the dog as measured with a radiolabled microsphere injection method. Arch Oral Bio11988;33:305-9. 6. Chahl L. Interactions of SP with putative mediators of inflammation and ATP. Eur J Pharmaco11977;44:45-9. 7. Lotz M, Vaughan J, Carson D. Effect of neuropeptides on production of inflammatory cytokines by human monocytes. Science 1988;241:1218-21. 8. Gazelius B, Edwall B, Olgart L, Lundberg M, Hokfelt T, Fischer J. Vasodilatory effects and coexistence of calcitonin gene-related peptide and substance P in sensory nerves of cat dental pulp. Acta Physiol Scand 1987; 130:33-40. 9. Kim S. Neurovascular interactions in the dental pulp in health and inflammation. J Endodon 1990; 16:48-53. 10. Saria A, Gamse R, Lundberg J, et el. Co-existence of tachykinins and calcitonin gene-related peptide in sensory nerves in relation to neurogenic inflammation. In: Haakinson R, Sundler F, eds. Tachykinin antagonists. Amsterdam: Elsevier Sciences Publications, 1985. 11. Byers M, Taylor P, Khayat B, Kimberly C. Effects of injury and inflammation on pulpal and periapical nerves. J Endodon 1990;16:78-84. 12. Khayat B, Byers M. Responses of nerve fibers to pulpal inflammation and periapical lesions in rat molars demonstrated by cacitonin gene-related peptide immunocytochemistry. J Endodon 1988;14:577-87. 13. Kimbedy C, Byers M. Inflammation of rat molar pulp and poriodontium causes increased calcitonin gene related peptide and axonal sprouting. Anat Rec 1988;222:289-300. 14. 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. 15. Ju G, Hokfelt T, Brodin E, et el. Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide-, and cholecystokinin-immunoreactive cells. Cell Tissue Res 1981 ;247:417-31. 16. Matsuyama T, Wanaka A, Yoneda S, et el. Two distinct calcitonin generelated poptide-containing peripheral nervous systems: distribution and quantitative difference between the iris and cerebral artery with specific reference to substance P. Brain Res 1986;373:205-12. 17. Zaidi M, Bevis P, Girgis S, Lynch C, Stevenso J, Maclntyre I. Circulating CGRP comes from the perivascular nerves. Eur J Pharmacol 1985;117: 283-4. 18. Heden P, Jernbeck J, Kjartansson J, Samuelson U. Increased skin flap survival and arterial dilation by calcitonin gene-related poptide. Scand J Plast Reconstr Surg 1989;23:11-6. 19. Kjartansson J, Dalsgaard C. Calcitonin gene-related peptide increases survival of a musculocutaneous critical flap in the rat. Eur J Pharmacol 1987; 142:355-8. 20. Kjartansson J, Dalsgaard C, Jonsson X. Decreased survival of experimental critical flaps in rats after sensory denervation with capsaicin. Plast Reconstr Surg 1987;79:218-221.