JOURNAL OF ENDODONTICS Copyright 2003 by The American Association of Endodontists

Printed in U.S.A. VOL. 29, NO. 4, APRIL 2003

Effect of an Intraosseous Injection of Depo-Medrol on Pulpal Concentrations of PGE2 and IL-8 in Untreated Irreversible Pulpitis
James Isett, DMD, MS, Al Reader, DDS, MS, Eric Gallatin, DDS, MS, Mike Beck, DDS, MA, and David Padgett, PhD

The purpose of this prospective, randomized, doubleblind study was to evaluate the pulpal concentrations of prostaglandin E2 (PGE2) and interleukin-8 (IL-8) in untreated teeth with irreversible pulpitis after the administration of an intraosseous injection of Depo-Medrol. Forty emergency patients with a clinical diagnosis of irreversible pulpitis experiencing moderate to severe pain participated. After receiving local anesthesia, patients randomly received, in a double-blind manner, an intraosseous injection of either 1 ml of Depo-Medrol (40 mg) (20 patients) or 1 ml of sterile saline placebo (control) (20 patients). No endodontic treatment was initiated. At 1 or 3 days after the intraosseous injection, the teeth were extracted and the pulpal tissue harvested. Prostaglandin E2 and interleukin-8 concentrations were determined by enzyme immunoassay. Results demonstrated a significantly (p < 0.05) lower concentration of prostaglandin E2 compared to the saline group at day 1. There were no significant (p > 0.05) differences between the two groups at day 3. The pulpal concentrations of prostaglandin E2 were reduced at 1 day after the intraosseous injection of Depo-Medrol.

A number of authors (13) have reported on inflammatory mediators in pulpal tissue and their modulation of the pain of pulpitis. Suppressing these mediators may help alleviate or control the pain of irreversible pulpitis. Corticosteroids are potent anti-inflammatory agents and inhibit the synthesis of cytokines by inducing the synthesis of antiinflammatory lipocortins (4). Hargreaves and Costello (5) demonstrated that the postoperative release of bradykinin was suppressed by corticosteroids. Boumpas et al. (6) reported that corticosteroids reduced capillary permeability, reduced neutrophil migration into tissue, reduced T-lymphocytes, and altered the macrophage production of cytokines including tumor necrosis factor and interleukins. Auphan et al. (7) discussed the role of nuclear factor kappa B (NF-kappa B) in activating many immuno-regulatory genes in
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response to pro-inflammatory stimuli. The inhibition of NFkappa B can be a major component of the anti-inflammatory activity of corticosteroids (7). Studies (8, 9) have demonstrated a reduction in neural sprouting and neuropeptide intensity of sensory nerves containing calcitonin gene related peptide or substance P when corticosteroids were administered. Gallatin et al. (10) demonstrated that the intraosseous injection of slow-releasing methylprednisolone (Depo-Medrol; Upjohn Co., Kalamazoo, MI) significantly reduced pain, percussion pain, and the use of analgesic medications for 7 days in teeth with untreated irreversible pulpitis. The purpose of using the Depo-Medrol was to temporarily reduce the patients symptoms until definitive endodontic treatment could be rendered. The authors speculated that the painful symptoms might be reduced due to the anti-inflammatory effects of the corticosteroid on the pulp. Two endogenous mediators have been associated with inflammation and the pain related to inflammation. Prostaglandin E2 (PGE2) has been documented to elicit a wide range of effects including vasodilation, increased vascular permeability, chemotaxis, and pain. Cohen (1) and Nakanishi et al. (2) found higher levels of PGE2 in painful teeth with irreversible pulpitis. The accumulation of neutrophilic leukocytes in a tissue, a defensive reaction to infection or injury, is a hallmark of inflammation. Interleukin-8 (IL-8) is a chemotactic and activating factor for human neutrophils (3). Various cells, such as monocytes, macrophages, and neutrophils, secrete IL-8 in response to pro-inflammatory agents (11). Haung et al. (3) found IL-8 production was increased in pulps diagnosed with irreversible pulpitis, whereas only negligible amounts of IL-8 were present in normal pulps. Therefore, PGE2 and IL-8 may play an important role in inflammation of the pulp. To date, no study has evaluated the effect of an intraosseous injection of Depo-Medrol, in untreated teeth with irreversible pulpitis, on the pulpal concentrations of PGE2 and IL-8. The purpose of this prospective, randomized, double blind study was to evaluate the pulpal concentrations of prostaglandin E2 and IL-8 in untreated teeth with irreversible pulpitis after the administration of an intraosseous injection of Depo-Medrol.

MATERIALS AND METHODS Pulpal tissues from 58 extracted teeth were used in this study. Twelve of these pulps were from either normal erupted third molars or from premolars extracted for orthodontic reasons (neg-

Vol. 29, No. 4, April 2003 TABLE 1. Initial values for Depo-Medrol and saline groups Value Age* Sex* Weight* Initial Pain Initial Percussion Pain Depo-Medrol 29 6.5 10 Females 10 Males 202 62.0 2.00 1.00 2.00 1.00 Saline 32 7.6 7 Females 13 Males 188 59.0 2.00 1.00 2.50 1.00 p 0.1966 0.3373 0.4753 0.7642 0.7352

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There were no significant differences between the two groups. * Mean SD. Median Interquartile Range; Ratings: 0 None, 1Mild, 2Moderate, 3Severe.

ative controls). Six pulps were from teeth with irreversible pulpitis (positive controls). None of these 18 teeth received Depo-Medrol or saline placebo injections. The 18 teeth were extracted at the initial appointment. These control pulps were used to analyze the levels of PGE2 and IL-8 in normal and inflamed pulps and used as controls for the enzyme-linked immunoassay. The other 40 pulps were harvested from 40 emergency patients who elected to have their teeth extracted. Forty adult patients presenting for emergency treatment participated in this study. All patients were in good health as determined by a written health history and oral questioning. Patients presenting with any contraindications to corticosteroids (systemic fungal infections, ocular herpes simplex, primary glaucoma, allergy to corticosteroids, ulcerative colitis, severe osteoporosis, poorly controlled insulin-dependent diabetes mellitus, a compromised immune status, and major psychosis) or any contraindications to the injection techniques or solutions were excluded from participation. The Ohio State University Human Subjects Review Committee approved this study, and written consent was obtained from each patient. Patients included in this study had a tooth with a clinical diagnosis of irreversible pulpitis and had actively spontaneous moderate to severe pain associated with the tooth. By definition, these teeth gave a positive response to an electric pulp tester (Kerr Pulp Tester, Analytic Technology, Redmond, WA) and a prolonged response to ice testing (Endo Ice; Hygenic Corporation, Akron, OH). The teeth also had percussion sensitivity, a history of spontaneous pain, and usually a radiographically widened periodontal ligament space. Patients with no response to pulp testing or periradicular pathosis (other than a widened periodontal ligament space) were excluded from the study. Various clinical and preoperative data were recorded for each patient (Table 1). Each patient was asked to rate their pain on a scale from zero to three. Zero indicated no pain. One indicated mild pain: pain that was recognizable but not discomforting. Two indicated moderate pain: pain that was discomforting but bearable. Three indicated severe pain: pain that caused considerable discomfort and was difficult to bear. Patients were asked to rate the pain to percussion using the same scale. Forty patients were given an intraosseous injection of either 1 ml (40 mg/ml) of Depo-Medrol or 1 ml of 0.9% preservative-free sterile saline (Elkins-Sinn Inc., Cherry Hill, NJ) placebo. The Depo-Medrol solution contained 40 mg/ml Depo-Medrol, 2.9% polyethylene glycol 3350 vehicle, 0.0195% myristyl-gamma-picolinium chloride preservative, and 0.9% sodium chloride. Another formulation of DepoMedrol contained benzyl alcohol and was not used. The solutions were placed in dental cartridges by removing the rubber plunger from the end of out-dated standard anesthetic cartridges. The cartridges were then emptied and washed, along with the rubber plungers, with soap and water and using a nylon bristle brush inside of the cartridge.

They were then rinsed with tap water and autoclaved. Using sterile technique, each sterilized anesthetic cartridge was filled with either 1.0 ml of Depo-Medrol or 1.0 ml of 0.9% preservative-free sterile saline solution, delivered with a tuberculin syringe, and the rubber stopper was replaced. The cartridges were wrapped with opaque tape and a corresponding four-digit random number was written on the tape to effectively blind the solutions. All patients were initially anesthetized with 3.6 ml of 2% lidocaine with 1:100,000 epinephrine (Astra Pharmaceutical Products, Inc., Westborough, MA) administered by nerve block or infiltration. The blinded solutions were administered via the intraosseous injection using the Stabident system (Fairfax Dental Inc., Miami, FL). The area of perforation was determined by the horizontal line of the buccal gingival margins of adjacent teeth and the vertical line that passed through the distal interdental papilla of the symptomatic tooth. The point approximately 2 mm below the intersection of these lines was the perforation site if the site was in attached gingiva. If the site was in alveolar mucosa, the perforation site was moved just above the junction of the attached gingiva and the alveolar mucosa on the same vertical line. When a distal perforation site could not be used (no interdental space), a mesial perforation site was selected. All patients received an infiltration injection of 0.1 ml of 2% lidocaine with 1:100,000 epinephrine at the intraosseous perforation site. The cortical plate was perforated using the Stabident perforator (a bevel-ended solid 27 gauge wire attached to a plastic hub) in a contra-angle, slow-speed handpiece. The perforator was placed through the gingiva and oriented perpendicular to the cortical plate. With the beveled end resting on the bone, the handpiece was activated in a series of short bursts, using light pressure, until a break through feeling was observed. If a break through feeling was not felt, the handpiece was activated again until the perforator was inserted to length. Before loading the cartridge into the syringe, the investigator vigorously shook the taped cartridge for 1 min. Because methylprednisolone acetate separates out of solution upon standing, shaking of each cartridge assured that, if the cartridge did contain methylprednisolone, the drug was back in solution and ready for injection. The cartridge was placed in a standard aspirating syringe and solution was deposited through the perforation site using a 27-gauge ultra-short needle with light pressure. The Depo-Medrol or saline was delivered into the cancellous space over a 2 min time period. If deposition required more than light pressure, the needle was rotated 90 degrees and the solution deposited. If solution could not be deposited (i.e. there was a large backflow of solution from the perforation site into the oral cavity), perforation was determined to be unsuccessful and the patient was eliminated from the study. No patients were eliminated for this reason. The senior author performed the perforations and all injections. No endodontic treatment was initiated. At the conclusion of the appointment, the patient was given a return appointment date for 1 or 3 days to have the tooth extracted and medications and a patient questionnaire to take home. Each patient received 20 600 mg tablets of ibuprofen (Motrin; H N Norton Co., Shreveport, LA). Patients were instructed to take the ibuprofen first for pain relief. Dosage was reviewed and labeled on the bottle as follows: take one tablet every 6 h as needed for pain. Patients were also given 20 tablets of acetaminophen with 30 milligrams of codeine (Tylenol #3; McNeil Pharmaceutical, Spring House, PA). Patients were instructed to take these tablets only if the ibuprofen did not sufficiently control their pain. The recommended dosage was given verbally and written on the vial as follows: take two tablets every 4 to 6 h as needed for pain. Patients

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were instructed to take only those medications given to them for this study. Patients were able to get additional pain medication, if needed, from the principal investigator. Each patient received a 1 to 3-day questionnaire (survey) to be completed every morning upon arising. They were instructed to record pain and percussion pain using the same four-point scale used for the initial pain recordings. Percussion pain was measured by asking the patient to tap on the affected tooth with their finger and rate the pain. Each patient recorded the number and type (ibuprofen or Tylenol #3) of pain medication taken each day. At the 1 or 3 day-scheduled appointment, the patient returned the daily surveys and all unused pain medication (to verify the amount of medication taken). All patients were anesthetized with 3.6 ml of 2% lidocaine with 1:100,000 epinephrine administered by nerve block or infiltration. If needed, patients then received an intraosseous injection using the Stabident system of 1.8 ml of 3% Mepivacaine HCL (Polocaine; Astra Pharmaceutical Products, Inc., Westborough, MA). The teeth were then extracted using accepted oral surgical procedures. After extraction, the teeth were immediately placed in liquid nitrogen. The teeth were grooved longitudinally and split with an elevator. The pulpal tissue was carefully removed from the tooth using a spoon excavator or explorer and placed in a cryogenic flask that was labeled with the four-digit random code number. Each pulpal sample was weighed to the nearest tenth of a milligram. The sample was then stored in liquid nitrogen until the assay procedure was started. The amount of PGE2 was analyzed using an enzyme immunoassay kit (EIA; Cayman Chemical Co., Ann Arbor, MI). The amount of IL-8 was analyzed using an enzyme-linked immunoassay kit (ELISA; Endogen, Inc., Woburn, MA). The amount of PGE2 and IL-8 in picograms per milligram of pulpal tissue was determined. Data were collected and statistically analyzed. The preoperative variables, for the two groups, were statistically analyzed using independent t tests (age, weight), the Fisher Exact test (sex), and the Mann-Whitney-Wilcoxon test (initial pain and percussion pain). Comparisons between the pulps of teeth with irreversible pulpitis and pulps of asymptomatic, normal teeth for the concentrations of PGE2 and IL-8 were analyzed using the Mann-WhitneyWilcoxon test. Comparisons between the Depo-Medrol and saline groups for the concentrations of PGE2 and IL-8, pain, percussion pain, and analgesic usage also were analyzed using the MannWhitney-Wilcoxon test.

FIG 1. The mean pulpal concentrations of PGE2 and IL-8 for the 18 control teeth (12 asymptomatic, normal teeth, and 6 inflamed teeth with irreversible pulpitis).

FIG 2. The mean pulpal concentrations of PGE2 and IL-8 for the Depo-Medrol and saline placebo groups at day 1.

RESULTS In the group of control teeth, which received neither DepoMedrol nor saline and were used only to establish baseline values, there were 18 teeth. For the 12 asymptomatic, normal teeth, the mean age was 25 yr and belonged to women. The initial mean pain and percussion ratings were none (0). For the 6 teeth diagnosed with irreversible pulpitis, the mean age was 29 yr, and 5 belonged to women and 1 belonged to a man. The initial mean pain rating was 2.5 1.0, and the initial mean percussion rating was 2.6 1.0. Fig. 1 shows the mean pulpal concentrations of PGE2 and IL-8 for the 18 control teeth. The PGE2 concentrations were 286 350 pg/mg for the 12 asymptomatic, normal teeth and 3493 4424 pg/mg for the irreversibly inflamed pulps. The IL-8 concentrations were 120 201 pg/mg for the normal pulps and 977 1467 pg/mg for the teeth with irreversible pulpitis. The concentrations of

PGE2 and IL-8 were significantly (p 0.01) higher in the pulps of teeth with irreversible pulpitis. In the experimental group of 40 teeth, twenty of the patients were in the Depo-Medrol group, and 20 patients were in the saline placebo group. The mean age of the patients was 28 yr with a range of 19 to 48 yr. Twenty-three of the patients were men, and 17 were women. There were 20 mandibular teeth and 20 maxillary teeth. Thirty-two of the teeth were molars, 8 were premolars, and none were anterior teeth. Table 1 shows the preoperative variables of age, weight, sex, initial pain and percussion pain for the two groups. No significant differences (p 0.05) were found between the two groups. Fig. 2 shows the mean pulpal concentrations of PGE2 and IL-8 for the Depo-Medrol and placebo groups at 1 day after the intraosseous injection. The PGE2 concentrations were 1,017 1236 pg/mg for the Depo-Medrol group and 6707 5823 pg/mg for the placebo group. The PGE2 concentration for the Depo-Medrol group was significantly (p 0.05) lower compared to the placebo group. The IL-8 concentrations were 575 786 pg/mg for the Depo-Medrol group and 2933 4284 pg/mg for the placebo group. The difference was not statistically significant (p 0.05). Fig. 3 shows the mean pulpal concentrations of PGE2 and IL-8 for the Depo-Medrol and placebo groups at 3 days after the intraosseous injection. The PGE2 concentrations were 2365

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(15). Opiates may regulate responses to IL-8 (16, 17). In theory, the analgesic medications taken during the study may have contributed to the lower levels of PGE2 and IL-8 on the third day. In conclusion, pulpal concentrations of PGE2 were reduced at 1 day after the intraosseous injection of 40 mg of Depo-Medrol in teeth with untreated irreversible pulpitis.
This study was supported by research funding from the Graduate Student Research Fund, Graduate Endodontics, The Ohio State University. This article was adapted from a thesis submitted by Dr. Isett in partial fulfillment of the requirements for the MS degree at The Ohio State University. A portion of this article was presented at the 57th Annual Session of the American Association of Endodontists, Honolulu, Hawaii. Dr. Isett, a former graduate student in endodontics, practices endodontics in private practice in York, PA.; Dr. Reader is a professor and the program director of graduate endodontics at The Ohio State University; Dr. Gallatin practices endodontics in private practice in Destin, FL; Dr. Beck is an associate professor for the Department of Oral Biology at The Ohio State University; and Dr. Padgett is an associate professor for the Department of Oral Biology at The Ohio State University. Address requests for reprints to Dr. Al Reader, Graduate Endodontics, College of Dentistry, The Ohio State University, 305 West 12 Avenue, Columbus, Ohio 43218.

FIG 3. The mean pulpal concentrations of PGE2 and IL-8 for the Depo-Medrol and saline placebo groups at day 3.

2922 pg/mg for the Depo-Medrol group and 2221 2038 pg/mg for the placebo group. The IL-8 concentrations were 676 913 pg/mg for the Depo-Medrol group and 899 823 pg/mg for the placebo group. There were no significant differences (p 0.05) between the two groups for either the PGE2 or IL-8. DISCUSSION The mean pulpal concentrations of PGE2 and IL-8 for the control teeth were statistically (p 0.01) higher for the teeth with irreversible pulpitis compared to asymptomatic teeth with normal pulps (Fig. 1). Our results are in agreement with previous authors. Both Cohen (1) and Nakanishi et al. (2) found higher concentrations of PGE2 in pulps of inflamed teeth. Huang et al. (3) demonstrated higher pulpal concentrations of IL-8 in teeth with irreversible pulpitis. Because our results agree with these previous authors, the methods and materials used in this study were probably valid for detecting quantitative levels of PGE2 and IL-8 in pulpal tissue. At day 1, the mean pulpal concentrations of PGE2 were statistically (p 0.05) reduced in the Depo-Medrol group compared to the saline group (Fig. 2). The results suggest that Depo-Medrol exerted an effect on pulpal inflammation on the first day. The mean pulpal concentration of IL-8 was also lower on the first day. Although not significant, the mean concentration of IL-8, for the saline group, was six times the concentration in the Depo-Medrol group (Fig. 2). A number of studies (1214) have shown that methylprednisolone is effective for IL-8 mediated inflammatory conditions. At day 3, the mean pulpal concentrations of PGE2 and IL-8 were similar between the two groups (Fig. 3), and there was no significant (p 0.05) difference between the Depo-Medrol and saline groups. Compared to day 1, the concentrations of PGE2 and IL-8 at day 3 may suggest that the anti-inflammatory effect of Depo-Medrol may be tapering by the third post-treatment day. However, Gallatin et al. (10) showed a clinical effect of DepoMedrol for 7 days in their study of irreversible pulpitis. If further research was carried out from day 3 to day 7, it may reveal an increase in concentrations of the inflammatory mediators, and, hence, a decrease in the effect of the Depo-Medrol. It is also possible that the anti-inflammatory effect of the analgesic medications may have lowered the concentrations of PGE2 and IL-8. Ibuprofen and acetaminophen may inhibit prostaglandin synthesis

References 1. Cohen JS, Reader A, Fertel R, Beck M, Meyers WJ. A radioimmunoassay determination of the concentrations of prostaglandins E2 and F2alpha in painful and asymptomatic human dental pulps. J Endodon 1985;11:330 5. 2. Nakanishi T, Matsuo T, Ebisu S. Quantitative analysis of immuonoglobulins and inflammatory factors in human pulpal blood from exposed pulps. J Endodon 1995;21:131 6. 3. Huang GT, Potente AP, Kim JW, Chugal N, Zhang X. Increased interleukin-8 expression in inflamed human dental pulps. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;88:214 20. 4. Minghetti L, Nicolini A, Polazzi E, et al. Down-regulation of microglial cyclo-oxygenase-2 and inducible nitric oxide synthase expression by lipocortin 1. Br J Pharmacol 1999;126:130714. 5. Hargreaves K, Costello A. Glucocorticoids suppress levels of immunoreactive bradykinin in inflamed tissue as evaluated by microdialysis probes. Clin Pharmacol Ther 1990;48:168 78. 6. Boumpas DT, Chrousos GP, Wilder RL, Cupps TR, Balow JE. Gluocorticoid therapy for immune-mediated diseases: Basic and clinical correlates. Ann Intern Med 1993;119:1198 208. 7. Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M. Immunosuppresion by glucocorticoids: Inhibition of NF-kappa B activity through induction of I kappa B synthesis. Science 1995;270:286 90. 8. Hong D, Byers MR, Oswald RJ. Dexamethasone treatment reduces sensory neuropeptides and nerve sprouting reactions in injured teeth. Pain 1993;55:171 81. 9. Ferrari AM, Byers MR. Chronic dexamethasone treatment and its effects on sensory neuropeptides, pulpal injury reactions and reparative dentin. Brain Res 1996;723:12534. 10. Gallatin E, Reader A, Nist R, Beck M. Pain reduction in untreated irreversible pulpitis using an intraosseous injection of Depo-Medrol. J Endodon 2000;26:633 8. 11. Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokines CXC and CC chemokines. Adv Immunol 1994;55: 97179 12. Youssef PP, Haynes DR, Triantafillou S, et al. Effects of pulse methylprednisolone on inflammatory mediators in peripheral blood, synovial fluid, and synovial membrane in rheumatoid arthritis. Arthritis Rheum 1997;40:1400 8. 13. Okawa K, Onda M, Miyashita M, Sasjima K. Systemic and pulmonary responses of inflammatory cytokine following esophagectomy. Nippon Ika Daigaku Zasshi 1998;65:429. 14. Kawamura T, Inada K, Okada K, Wakusawa R. Methylprednisolone inhibits the increase of interleukin 8 and 6 during open heart surgery. Can J Anaesth 1995;42:399 403. 15. Vane JR. Inhibition of prostaglandins synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol 1971;231:2325. 16. Grimm MC, Ben-Baruch A, Taub DD, et al. Opiates transdeactivate chemokine receptors: Delta and mu opiate receptor-mediated heterologous desensitization. J Exp Med 1998;188:31725. 17. Grimm MC, Ben-Baruch A, Taub DD, et al. Opiate inhibition of chemokine-induced chemotaxis. Ann NY Acad Sci 1998;840:9 20