Basic Research—Biology

Localization of 14C-Labeled 2% Lidocaine Hydrochlorideafter Intraosseous Anesthesia in the RabbitTakashi Goto, DDS, PhD, Hideki Mamiya, DDS, PhD, Tatsuya Ichinohe, DDS, PhD,and Yuzuru Kaneko, DDS, PhD

Abstract

Objective: The purpose of this study was to investigatethe tissue distribution of lidocaine hydrochloride inmandibular bone marrow after intraosseous anesthesia(IOA) in rabbits.Methods:Weusedmacroautoradiogra-phy to examine the tissue distribution of a 14C-labeled2% lidocaine hydrochloride solution containing1:80,000 epinephrine (14C-lidocaine). Under generalanesthesia, 14C-lidocaine was injected intraosseouslyor paraperiosteally. After IOA, animals were dividedinto three groups and observed at 1 (IOA-1), 5 (IOA-5),and 10 minutes (IOA-10) after injection. After infiltrationanesthesia (IA), animals were observed at 1 minute afterinjection. Results: The accumulation of 14C-lidocainewasobserved around the injection site in both the IA and theIOA groups. Paraperiosteally injected 14C-lidocainediffused to the surrounding tissues such as the lip,whereas IOA showed concentrated accumulation aroundthe root apex throughout the experiment. The distributionarea was significantly smaller in the IOA-1 group than inthe IA group. The distribution area in the IOA-5 groupwas larger than those in the IOA-1 and IOA-10 groups.Conclusions: The accumulation of 14C-lidocaine injectedby IOA in rabbits was concentrated around the root apex.These results may explain the rapid onset time of IOA. (JEndod 2011;37:1376–1379)

Key WordsAlveolar bone, intraosseous anesthesia, lidocaine,macroautoradiography, radioisotope, tissue distribution

From the Department of Dental Anesthesiology, TokyoDental College, Chiba-shi, Japan.

Address requests for reprints to Dr Takashi Goto,Department of Dental Anesthesiology, Tokyo Dental College,1-2-2, Masago, Mihama-ku, Chiba-shi, 261-0011, Japan.E-mail address: gotoutakashi@tdc.ac.jp0099-2399/$ - see front matter

Copyright ª 2011 American Association of Endodontists.doi:10.1016/j.joen.2011.05.039

1376 Goto et al.

Local anesthesia is essential for pain control during dental treatment. Currently,infiltration anesthesia (IA) is the most common procedure. However, an anesthetic

effect in the mandibular molars is frequently insufficient after IA only (1, 2). In thesecases, supplemental techniques such as intraosseous anesthesia (IOA), intraligamentalanesthesia, and/or block anesthesia are combined with IA. In general, IOA is used aseither a primary injection technique or a supplemental injection technique. Of those,IOA is often used as a supplemental technique after a failed inferior alveolar nerveblock, and this technique provides fast and sufficient anesthesia even in themandibular molars (3–6). In a rabbit study, the onset time, which was defined asthe elapsed time until the electromyogram of the digastric muscle disappeared afterthe injection, was reported less than 1 minute after primary IOA (7). In human studies,it is also reported that the onset time after primary IOA was quite rapid (8–15). Theanesthetic area and duration of anesthesia after supplemental IOA were sufficient forthe treatment even in the tooth with pulpitis (3–6). Primary IOA also has advantagesof minimal anesthetic effects beyond the intended site compared with an alveolarnerve block (13–15). Many studies have investigated the clinical findings such asthe onset or duration and anesthetic efficacy after local anesthetics (8–12, 15).However, there are few studies that investigated the tissue distribution of ananesthetic solution after local anesthesia (16–19). Yamazaki et al (16) reported thediffusion process of 14C-labeled 2% lidocaine hydrochloride in rabbit oral tissue afterIA. However, there has been no study on the tissue distribution of a local anestheticsolution in the bone marrow administered as IOA. Therefore, we used macroautora-diography to investigate how anesthetic solution administered by primary IOA diffusionin the oral tissues.

Materials and MethodsThis study was conducted in compliance with the Guidelines for the Treatment of

Experimental Animals at Tokyo Dental College in accordance with the guidelines of theJapanese government (approval no. 222502). Thirty-six male JapanWhite rabbits, 10 to12 weeks old, weighing between 2.5 and 3.0 kg, were used in this study. The rabbitswere anesthetized with 3% isoflurane. Tracheotomy was performed under local anes-thesia, and then an 18- to 20-French size pediatric endotracheal tube was inserted intothe trachea and fixed. A venous indwelling catheter was inserted into the left auricularmarginal vein as a route for fluid infusion and drug administration. Anesthesia wasmaintained by an intermittent intravenous injection of sodium thiopental (Ravonal;Mitsubishi Tanabe Pharma Corporation, Kyoto, Japan).

14C was used as a labeled radioisotope (RI) because it had an appropriate half-lifelong enough for this study, and it was commercially available for labeled lidocaine. Theexperimental local anesthetic solution consisted of 14C-labeled 2% lidocaine hydro-chloride (American Radiolabeled Chemicals Inc, St. Louis, MO) containing 1:80,000epinephrine (14C-lidocaine). The concentration of radioactivity was 37.2 kBq/mL.

The 14C-lidocaine was injected by IOA or IA. In the IOA groups, the tip of the needlewas placed in the vicinity of the root apex of the lower right incisor. The X-tip system(Dentsply International Inc, York, PA) was used for IOA. The diameter of the guide sleevewas 0.64 mm. The length of the guide sleeve and drill were modified to 4 and 6 mm,respectively. The X-tip was inserted through the lateral compact bone near the inferiorborder of the mandible through the skin by using a slow-speed handpiece at 20,000rpm. The 14C-lidocaine was injected through the guide sleeve of the X-tip by using

JOE — Volume 37, Number 10, October 2011

Basic Research—Biology

a 1-mL syringe with a 27-G needle. The injection volume was 0.03 mL. Ineight rabbits, a dental cone-beam computed tomography scan was usedimmediately after an insertion of the guide sleeve to confirm that the tip ofthe needle was within the mandible. In the IA group, 14C-lidocaine wasinjected into the vestibular fornix corresponding to the right incisor.The injection needle was inserted to a depth of 5 mm in an apical direc-tion from the mucous membrane side. A volume of 0.04 mL was injectedusing amicrosyringe with a 30-G needle. After the IOA with 14C-lidocaine,animals were divided into three groups. Observation was preformed at 1(IOA-1 group, n = 9), 5 (IOA-5 group, n = 9), and 10 minutes (IOA-10group, n = 9) after injection. These time periods were determined basedon the previous study (17). After the IA injection with 14C-lidocaine,observation was performed at 1 minute after injection (IA group, n =9). Each rabbit received one anesthesia in this study.

After the elapse of each observation time, animals were sacrificedwith an overdose of sodium thiopental. The animals were rapidly frozenwith liquid nitrogen, and then the maxilla and mandible were excised inone piece. These samples were embedded in 8% carboxymethylcellu-lose paste, frozen with acetone, cooled in dry ice, and prepared as spec-imen blocks. Sections of 50-mm thickness were prepared using anautocryotome from the specimen blocks. Sections were made parallelto the plane perpendicular to the guide sleeve direction. This planewas almost parallel to the plane that includes the long axis of the incisor.

Figure 1. Specimen and accumulation image are superimposed and accumulatioaccumulation image. Superposition of these two images identifies the location whthe volume of 14C-lidocaine (control site); b, the measurement area of the volume1, 1 minute after IA; 2, 1 minute after IOA; 3, 5 minutes after IOA; 4, 10 minutes

JOE — Volume 37, Number 10, October 2011

The sections were dried in a freezer at�20�C for about 10 days. Then,the section was contacted with an imaging plate for 90 minutes in animaging cassette. Accumulation imageswere acquiredwith a BioimagingAnalyzer System (BAS; Fuji Film, Tokyo, Japan). BAS is a device that visu-alizes radioisotopic information on the imaging plate. Specimens werescanned, and anatomic imaging data were obtained. Finally, twoimaging data were superimposed, and the position of the image showingaccumulation was determined (Fig. 1A).

Images obtained from the BAS were analyzed with image analysissoftware (ImageJ 1.42q; National Institutes of Health, Bethesda, MD).A circular region of interest with a diameter of 45 pixels (pixel size= 200 mm) was set at the center of the local anesthetic injection site.The volume of the 14C-lidocaine was quantified by determining theamount of radioactivity within the circle, calculated as density � pixelcount, and the volume was compared with that at the control site.Because a radiation dosage obtained from the BAS was in proportionto pixels, the RI level, and the volume of 14C-lidocaine, the radiationdosage obtained from the BAS was considered as volume of the14C-lidocaine because pixels and RI level were constant in this study(Fig. 1Aa and Ab). To investigate the time course change in diffusionarea after IOA, regions of 14C accumulation in images obtained wereselected, and the area of local anesthetic distribution (pixel count)was calculated.

n area is determined. (A) Superposition of the prepared specimen and theere 14C-lidocaine is present in rabbit oral tissue. a, the measurement area ofof 14C-lidocaine (injection site). (B) The distribution area of 14C-lidocaine.

after IOA.

Localization of Lidocaine after Intraosseous Anesthesia 1377

Figure 3. The distribution area in each group. The distribution area in theIOA-1 group was smaller than that in the IA group. The distribution area inthe IOA-5 group was larger than those in the IOA-1 and IOA-10 groups.Data were expressed as mean � standard deviation. *A statistically significantdifference (P < .05). IA, 1 minute after infiltration anesthesia; IOA-1, 1 minuteafter intraosseous anesthesia; IOA-5, 5 minutes after intraosseous anesthesia;IOA-10, 10 minutes after intraosseous anesthesia.

Basic Research—Biology

All measurement results are presented as mean � standard

deviation. Statistical analysis of the volume of 14C-lidocaine and thedistribution area was performed using the Student’s t test for pairedsamples and non–repeated-measures analysis of variance, respectively.Multiple comparison testing was performed by the Student-Newman-Keuls test, with a critical level of less than 5% considered significant.

ResultsThe accumulation of 14C-lidocaine injected into the oral tissues

was observed around the injection site in both groups. The 14C-lidocaineinjected as IA had diffused to the surrounding tissues such as the lip,whereas 14C-lidocaine injected as IOA showed minimal diffusion tothe surrounding tissues (Fig. 1B). The volume of 14C-lidocaine at theinjection site was greater than in the control site in both groups. Notime course change was observed among IOA-1, IOA-5, and IOA-10groups (Fig. 2).

The IA group showed no accumulation of 14C-lidocaine aroundthe root apex. The IOA-1 group showed accumulation of 14C-lidocainearound the root apex. The distribution area was smaller in the IOA-1group than that in the IA group (Fig. 3). The IOA group showed accu-mulation of the local anesthetic around the root apex at 1, 5, and 10minutes after the injection. The distribution area in the IOA-5 groupwas larger than those in the IOA-1 and IOA-10 groups (Fig. 3).

DiscussionIt is reported that the onset time was 10 to 12 seconds (14), 1.64

� 1.95 minutes (12) in human studies, and 0.6� 0.1 minutes (7) inrabbit studies after IOA. In our study in the IOA group, 14C-lidocaine hadalready reached the apical area 1 minute after injection. In contrast,distribution of 14C-lidocaine in the IA group was limited to the injectionsite and did not extend to the apical area 1 minute after injection.Yamazaki et al (16) reported that IA required 5minutes for the diffusionof 14C-lidocaine around the root apex using the same method with thisstudy. Other studies (12, 20, 21) showed that IA requires 5 to 10minutes for effective anesthesia.

Time course observation of the distribution area after IOA showedthat the distribution was greater in the IOA-5 group than those in theIOA-1 and IOA-10 groups. The reason for the greater area in theIOA-5 group may be a continuous spread of 14C-lidocaine into the

Figure 2. The volume of 14C-lidocaine in the injection site and the controlsite. In both groups, the volume of 14C-lidocaine at the injection site wasgreater than that in the control site. Data were expressed as mean � standarddeviation. *A statistically significant difference (P < .05).

1378 Goto et al.

surrounding tissues. The smaller area in the IOA-10 group might becaused by the abundant blood stream in the mandibular bone marrowand a consequently greater removal of the 14C-lidocaine by bloodstream compared with that in the IOA-5 group, even if 14C-lidocaineremained at the injection site 10 minutes after injection.

The reported duration of anesthesia, which was defined as theelapsed time until the electromyography of digastric muscle amplituderecovered to 50% of the control value after the IOA, was 51.6 � 9.4minutes in a rabbit study (7). In a human study using lateral incisor,the duration of anesthesia was 26 � 13.3 (12). These results suggestthat 14C-lidocaine can remain in the apical area even more than 10minutes after injection. No time course change of the volume of14C-lidocaine around the root apex in the IOA groups suggests theconcentrated accumulation around the root apex in the IOA groups.This may be in part evidential for this duration of anesthesia. This studyshowed minimal diffusion to the surrounding tissues when IOA wasperformed. Baker et al (22) reported that gingival bleeding duringflap reflection was greater in the IOA group than in the IA group, whichsuggested that local anesthetics administered by IOA might minimallyreach the gingiva.

There are some reports on the effect of epinephrine on dental pulpischemia (23, 24). However, there had been no report on pulp necrosisafter IOA. Clinical studies (9–11) have shown no effect on the pulp afterintraosseous injections. All pulps tested at normal baseline levels atfollow-up visits.

We used 0.03 mL of local anesthetic solution in the IOA groupsbased on the previous study by Ito et al (7). Meanwhile, we used 0.04mL of local anesthetic solution in the IA group based on our pilot study.Although Yamazaki et al (16) used 0.06 mL for the IA, we confirmed thatthe smaller dosage produced a clearer image in the imaging plate in ourpilot study. However, we could not obtain a clear enough image with 0.03mL compared with 0.04mL for the IA. Because the IOA dosage was equalto or smaller than that of IA in the clinical settings (12, 25), the differencebetween IOA dosage and IA dosage in this study may be of little

JOE — Volume 37, Number 10, October 2011

Basic Research—Biology

importance. Because we observed only the two-dimensional distributioncondition, three-dimensional distribution using horizontal and serialsections needs to be observed in a future study. In addition, studies onthe distribution of anesthetic solutions over a 30- to 60-minute timeperiod should be performed. This might be a part of evidence for timecourse change of the anesthetic efficacy after IOA.

ConclusionThe accumulation of 14C-lidocaine after IA and IOA was confirmed

in rabbits. Oneminute after IOA, 14C-lidocaine was detected at the apicalarea. The 14C-lidocaine injected as IA had diffused to the surroundingtissues such as the lip, whereas 14C-lidocaine injected as IOA showedminimal diffusion to the surrounding tissues. The accumulation of14C-lidocaine injected as IOA in rabbits was concentrated around theroot apex.

AcknowledgmentsThe authors thank Takakai Yamazaki (Department of Ultra-

structual Science, Tokyo Dental College, Tokyo, Japan) for his directguidance and revisions to the manuscript, Tsukasa Sano andKeiichi Nishikawa (Department of Oral and MaxillofacialRadiology, Tokyo Dental College) for their advice, and Drs ToshiakiArima (Tokyo Nuclear Services Co, Ltd, Tokyo, Japan) and KazunoriAnzai (National Institute of Radiological Sciences, Chiba, Japan)for their guidance.

The authors deny any conflicts of interest related to this study.

References1. Wallace JA, Michanowicz AE, Mundell RD, Wilson EG. A pilot study of the clinical

problem of regionally anesthetizing the pulp of an acutely inflamed mandibularmolar. Oral Surg Oral Med Oral Pathol 1985;59:517–21.

2. Brown BD. The failure of local anesthesia in acute inflammation. Some recentconcepts. Br Dent J 1981;151:47–51.

3. Nusstein J, Kennedy S, Reader A, Beck M, Weaver J. Anesthetic efficacy of the supple-mental X-tip intraosseous injection in patients with irreversible pulpitis. J Endod2003;29:724–8.

4. Nusstein J, Reader A, Nist R, Beck M, Meyers WJ. Anesthetic efficacy of thesupplemental intraosseous injection of 2% lidocaine with 1:100,000 epinephrinein irreversible pulpitis. J Endod 1998;24:487–91.

5. Parente SA, Andreson RW, Herman WW, Kimbrough WF, Weller RN. Anestheticefficacy of the supplemental intraosseous injection for tooth with irreversiblepulpitis. J Endod 1998;24:826–8.

JOE — Volume 37, Number 10, October 2011

6. Reisman D, Reader A, Nist R, Beck M, Weaver J. Anesthetic efficacy of the supple-mental intraosseous injection of 3% mepivacaine in irreversible pulpitis. Oral SurgOral Med Oral Pathol Oral Radiol Endod 1997;84:676–82.

7. Ito E, Ichinohe T, Shibukawa Y, Aida H, Kaneko Y. Anesthetic duration of lidocainewith 10% dextran is comparable to lidocaine with 1:160,000 epinephrine afterintraosseous injection in the rabbit. Oral Surg Oral Med Oral Pathol Oral RadiolEndod 2007;104:e26–31.

8. Remmers T, Glickman G, Spears R, He J. The efficacy of IntraFlow intraosseousinjection as a primary anesthesia technique. J Endod 2008;34:280–3.

9. Coggins R, Reader A, Nist R, Beck M, Meyers WJ. Anesthetic efficacy of the intraoss-eous injection in maxillary and mandibular teeth. Oral Surg Oral Med Oral PatholOral Radiol Endod 1996;81:634–41.

10. Replogle K, Reader A, Nist R, Beck M, Weaver J, Meyers WJ. Anesthetic efficacy of theintraosseous injection of 2% lidocaine (1:100,000 epinephrine) and 3% mepiva-caine in mandibular first molars. Oral Surg Oral Med Oral Pathol Oral Radiol Endod1997;83:30–7.

11. Gallatin J, Reader A, Nusstein J, Beck M, Weaver J. A comparison of two intraosseousanesthetic techniques in mandibular posterior teeth. J Am Dent Assoc 2003;134:1476–84.

12. Nusstein J, Wood M, Reader A, Beck M, Weaver J. Comparison of the degree ofpulpal anesthesia achieved with the intraosseous injection and infiltration injectionusing 2% lidocaine with 1:100,000 epinephrine. Gen Dent 2005;53:50–3.

13. Lilienthal B. A clinical appraisal of intraosseous dental anesthesia. Oral Surg OralMed Oral Pathol 1975;39:692–7.

14. Leonard MS. The efficacy of an intraosseous injection system of delivering localanesthetic. J Am Dent Assoc 1995;126:81–6.

15. Brown R. Intraosseous anesthesia: a review. J Calif Dent Assoc 1999;27:785–92.16. Yamazaki T, Mamiya H, Ichinohe T, Kaneko Y. Distribution of lidocaine in alveolar

tissues in rabbits. J Hard Tissue Biol 2009;18:95–100.17. Pettersson LO, Akerman B. Influence of hyaluronidase upon local infiltration anes-

thesia by lidocaine. Scand J Plast Reconstr Surg 1984;18:297–301.18. Lewis-Smith PA. Adjunctive use of hyaluronidase in local anaesthesia. Br J Plast Surg

1986;39:554–8.19. Moorthy SS, Dierdorf SF, Yaw PB. Influence of volume on the spread of local

anesthetic-methylene blue solution after injection for intercostals block. AnesthAnalg 1992;75:389–91.

20. Miyoshi T, Aida H, Kaneko Y. Comparative study on anesthetic potency of dentallocal anesthetics assessed by the jaw-opening reflex in rabbits. Anesth Prog 2000;47:35–41.

21. Ohkado S, Ichinohe T, Kaneko Y. Comparative study on anesthetic potency depend-ing on concentrations of lidocaine and epinephrine; assessment of dental localanesthetic using the jaw-opening reflex. Anesth Prog 2001;48:16–20.

22. Baker TF, Torabinejad M, Scbwartz SF, Wolf D. Effect of intraosseous anesthesia oncontrol of hemostasis in pigs. J Endod 2009;35:1543–5.

23. Kim S. Ligamental injection: a physiological explanation of its efficacy. J Endod 1986;12:486–91.

24. Ahn J, Pogrel MA. The effects of 2% lidocaine with 1:100,000 epinephrine on pulpaland gingival blood flow. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:197–202.

25. Kleber CH. Intraosseous anesthesia Implications: instrumentation and techniques.J Am Dent Assoc 2003;134:487–91.

Localization of Lidocaine after Intraosseous Anesthesia 1379