This study was approved by the Animal Experiment Committee of the Nippon Dental University School of Life Dentistry (approval No. 10-29) and was conducted in accordance with the guidelines laid down by ARRIVE (Animal Research: Reporting of in vivo Experiments).
Animals
One hundred and twenty-eight 7–8-week-old pathogen-free Wistar male rats (Tokyo Laboratory Animals Science Co., Ltd., Tokyo, Japan) were used for this study. We divided the rats into two groups: the control group administered with lidocaine only, and the treatment group administered with lidocaine with epinephrine. Each group included 64 animals. Two rats per cage were housed in the animal room of the University’s isotope facility and were given unrestricted access to water and food.
Formulation of test drugs
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(a)
Lidocaine
We dissolved 3.5 g of carboxymethyl cellulose sodium salt (CMC) in 100 mL of 2% lidocaine hydrochloride solution to formulate a 2% lidocaine hydrochloride gel.
Then, we added 25 µL of 14C-labeled 2% lidocaine hydrochloride (American Radiolabeled Chemicals, Inc., St. Louis, MO, USA) and 5 µL of 0.9% sodium chloride to 500 µL of 2% lidocaine hydrochloride gel.
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(b)
Lidocaine with epinephrine
We added 5 µL of 1 mg/mL epinephrine in place of 5 µL of 0.9% NaCl as used in (a).
Indigo carmine dye was added to both test drugs to aid visibility in order to excise the correct area of maxillary tissue.
Lidocaine measurement
Lidocaine was measured according to the method described by Akimoto et al. [18].
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(a)
Sample collection
Rats were administered pentobarbital (50 mg/kg) intraperitoneally for inducing sleep and were placed on their backs. A 5 µL dose of both the test drugs was applied to the oral mucosae of the animals of corresponding test groups using an applicator tip with an internal diameter of 2 mm and a length of 4 mm. This drug application was at the intersection of the midline of the palate and a line joining the centers of the bilateral second molars (Fig. 1). Samples of the maxilla and oral mucosa were collected from 100 rats, serum was collected from 24 rats, and autoradiographs were recorded for 4 rats.
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(i)
Maxillary tissue
One hundred rats (50 rats from each study group) were used for tissue measurement. A total of 10 time points were used in the study: 0.5, 2, 4, 7, 10, 20, 30, 40, 50, and 60 min after application. Sleeping rats were decapitated with a guillotine and the gel remaining on the surface of the mucosa was removed with cotton swabs. Thus, there were 5 samples at each time point. The drug-applied region was excised from the upper jaw using bone scissors, and the mucosa was separated from the bone using bone forceps. These samples of the mucosa and bone were minced with the bone scissors for radioactivity measurement.
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(ii)
Serum
Another 24 rats (12 rats from each study group) were used for serum evaluation. At 0.5, 2, 5, 10, 20, 30, 40, 50, and 60 min after application, 0.4 mL blood was collected from the left femoral artery and centrifuged at 4 °C and 15,000×g for 20 min to obtain the serum. Thus, 12 blood samples for each time point were collected from each rat of both groups. The study animals were euthanized by intraperitoneal administration of 150 mg/kg pentobarbital sodium after sample collection.
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(b)
Radioactivity measurements
The collected samples of the maxillary tissue (10–50 mg) or serum (50 µL) were placed in a liquid scintillation counter vial, and 0.5 mL of tissue solubilizer (Solvable®; PerkinElmer, Waltham, MA, USA) was added. This mixture was warmed and agitated at 60 °C for 2 h, and 25 µL of acetic acid was added to neutralize it.
To this solution, a liquid scintillation cocktail (AQUASOL-2®; PerkinElmer, Waltham, MA, USA) was added, and the resulting solution was left in the dark for 24 h. Thereafter, the radioactivity (dpm) was measured using a liquid scintillation counter (LEC-6100; Aloka, Tokyo, Japan).
The amount of lidocaine in the mucosa or palatine bone was calculated per wet weight of tissue (ng/mg wet weight) from the measured value and specific activity. The amount of lidocaine in the serum was indicated in terms of 14C-radioactivity (dpm/mL).
Autoradiography observations
Autoradiography was conducted according to the method described by Hashimoto et al. [19].
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(a)
Section preparation
The remaining 4 rats (2 rats from each study group of lidocaine and lidocaine with epinephrine) were used for autoradiography. The same method of lidocaine measurement was followed as in the previous section. The maxilla was removed 10 and 40 min after lidocaine or lidocaine with epinephrine application, embedded in CMC, and placed on hexane dry ice to prepare frozen maxillary tissue blocks. A cryomicrotome (CM4050S®; Leica Microsystems, Wetzlar, Germany) was then used to prepare coronal sections of 10 µm thickness. These sections were placed on an adhesive sheet (Transfer Film®; Leica Microsystems, Wetzlar, Germany) and dried.
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(b)
Film observations
The dried sections were pressed onto an x-ray film (BioMax® XAR Film; Kodak, Rochester, NY, USA) and exposed to a temperature of − 80 C for 40 days. The developed films were placed on sections stained with 0.25% eosin (EosinY®; Nacalai Tesque Inc., Kyoto, Japan), and the radioactive isotope distribution was observed using a transmission scanner (GT9500®; EPSON, Nagano, Japan).
Statistical analyses
Measurements are indicated as means ± standard deviations. Measurements at each time point were compared between the two groups using an unpaired t-test or Welch’s t-test if unequal variance was observed, with p < 0.05 regarded as significant. A software was used for statistical analyses (IBM SPSS® Statistics ver. 25; IBM Japan Ltd., Tokyo, Japan).