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Immunohistochemical Analysis of Nerve Distribution in Mandible of Rats
Kanae KudoDDS, PhD,
Katsunori TanakaDDS, PhD,
Kimiharu AmbeDDS, PhD,
Hiroyoshi KawaaiDDS, PhD, and
Shinya YamazakiDDS, PhD
Article Category: Research Article
Volume/Issue: Volume 66: Issue 2
Online Publication Date: Jan 01, 2019
DOI: 10.2344/anpr-66-01-10
Page Range: 87 – 93

problems exist with the use of the normal human mandible for tissue specimens; therefore, the rat mandible was used in the present study, and the nerve distribution density was analyzed. The rat mandible has been frequently used as an experimental model imitating the human mandible for anatomical, histological, and surgical studies. In addition, it has been demonstrated as a highly reproducible ideal histologic model. 14 , 15 Therefore, the nerve distribution density in the human mandible can be analogized from these results to a certain degree

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Figure 1. ; The region was divided vertically into 5 sections (A, B, C, D, and E). In addition, the C region was divided horizontally into equal thirds (CL, CC, and CP). Nerve distribution density was measured in each region, and the number of nerves per bone area was expressed as number of nerve fibers per mm2.
Kanae Kudo,
Katsunori Tanaka,
Kimiharu Ambe,
Hiroyoshi Kawaai, and
Shinya Yamazaki
<bold>Figure 1.</bold>
Figure 1.

The region was divided vertically into 5 sections (A, B, C, D, and E). In addition, the C region was divided horizontally into equal thirds (CL, CC, and CP). Nerve distribution density was measured in each region, and the number of nerves per bone area was expressed as number of nerve fibers per mm2.

Figure 2. ; Protein gene product (PGP)- and calcitonin gene-related peptide (CGRP)-positive nerve to measure the nerve distribution. The bone area in each region was measured, and the number of nerves in each bone area was measured using AxioVision image analysis software. Red arrows indicate nerves. The scale bar shows 50 μm.
Kanae Kudo,
Katsunori Tanaka,
Kimiharu Ambe,
Hiroyoshi Kawaai, and
Shinya Yamazaki
<bold>Figure 2.</bold>
Figure 2.

Protein gene product (PGP)- and calcitonin gene-related peptide (CGRP)-positive nerve to measure the nerve distribution. The bone area in each region was measured, and the number of nerves in each bone area was measured using AxioVision image analysis software. Red arrows indicate nerves. The scale bar shows 50 μm.

Figure 3. ; Statistical analysis of vertical nerve distribution density. All graphs are shown in mean ± standard deviation. PGP: All nerves were stained by protein gene product 9.5 antibody. CGRP: Pain-sensitive nerves were stained by calcitonin gene-related peptide antibody. *p < .05; **p < .01; ns, no significant difference on chi-square test.
Kanae Kudo,
Katsunori Tanaka,
Kimiharu Ambe,
Hiroyoshi Kawaai, and
Shinya Yamazaki
<bold>Figure 3.</bold>
Figure 3.

Statistical analysis of vertical nerve distribution density. All graphs are shown in mean ± standard deviation. PGP: All nerves were stained by protein gene product 9.5 antibody. CGRP: Pain-sensitive nerves were stained by calcitonin gene-related peptide antibody. *p < .05; **p < .01; ns, no significant difference on chi-square test.

Figure 4. ; Statistical analysis of horizontal nerve distribution density. All graphs are shown as mean ± standard deviation. PGP: All nerves were stained by protein gene product 9.5 antibody. CGRP: Pain-sensitive nerves were stained by calcitonin gene-related peptide antibody. *p < .05; **p < .01; ns, no significant difference by chi-square test.
Kanae Kudo,
Katsunori Tanaka,
Kimiharu Ambe,
Hiroyoshi Kawaai, and
Shinya Yamazaki
<bold>Figure 4.</bold>
Figure 4.

Statistical analysis of horizontal nerve distribution density. All graphs are shown as mean ± standard deviation. PGP: All nerves were stained by protein gene product 9.5 antibody. CGRP: Pain-sensitive nerves were stained by calcitonin gene-related peptide antibody. *p < .05; **p < .01; ns, no significant difference by chi-square test.

Figure 7 ; Effect of ropivacaine on the blood flow rate of rat upper lip. After 20 μL of 0.9% NaCl (○), 0.5% ropivacaine (•), 10 μg/mL epinephrine (△), or ropivacaine with epinephrine (▴) was injected to the rat upper left lip, the labial blood flow rate was measured by a laser Doppler flow meter (ALF21, ADVANCE) with a contact-type probe. Data are mean ± SD (n = 4). *P < .05 epinepherine versus ropivacaine with epinephrine.
Mikiko Yamashiro,
Shuichi Hashimoto,
Asako Yasuda, and
Katsuhisa Sunada
<bold>Figure 7</bold>
Figure 7

Effect of ropivacaine on the blood flow rate of rat upper lip. After 20 μL of 0.9% NaCl (○), 0.5% ropivacaine (•), 10 μg/mL epinephrine (△), or ropivacaine with epinephrine (▴) was injected to the rat upper left lip, the labial blood flow rate was measured by a laser Doppler flow meter (ALF21, ADVANCE) with a contact-type probe. Data are mean ± SD (n = 4). *P < .05 epinepherine versus ropivacaine with epinephrine.

Figure 1. ; Schematic of the experimental protocol. Each rat was placed in a wire net container on a heated glass base 10 minutes prior to the start of the experiment. Baseline values were recorded 5 minutes prior to subcutaneous injection. Paw withdrawal latency was monitored immediately after the injection of the test solution; latency was then monitored every 5 minutes thereafter for 40 minutes.
Yukako Tsutsui and
Katsuhisa Sunada
Figure 1. 
Figure 1. 

Schematic of the experimental protocol. Each rat was placed in a wire net container on a heated glass base 10 minutes prior to the start of the experiment. Baseline values were recorded 5 minutes prior to subcutaneous injection. Paw withdrawal latency was monitored immediately after the injection of the test solution; latency was then monitored every 5 minutes thereafter for 40 minutes.

Figure 1 ; Measurement of the blood pressure (BP) and heart rate (HR) of rats using a sphygmomanometer. The rats were warmed to 37°C to 38°C using a cylinder thermostat and placed in a restrainer. The sphygmomanometer cuff was placed around the tail of each rat to measure the BP and HR.
Yukako Tsutsui and
Katsuhisa Sunada
<bold>Figure 1</bold>
Figure 1

Measurement of the blood pressure (BP) and heart rate (HR) of rats using a sphygmomanometer. The rats were warmed to 37°C to 38°C using a cylinder thermostat and placed in a restrainer. The sphygmomanometer cuff was placed around the tail of each rat to measure the BP and HR.

Figure 6. ; Effect on CAPs of IDA (n = 6) This graph shows an effect of different concentrations of IDA on the amplitude of CAPs. Reductions in the amplitude of the rat sciatic nerve by 3 different concentrations of IDA, measured 5 minutes after its application. The concentrations of IDA were 16 μM, 80 μM and 160 μM. The amplitude of the CAPs was unchanged by the administration of the IDA.
<bold>Figure 6.</bold>
Figure 6.

Effect on CAPs of IDA (n = 6)

This graph shows an effect of different concentrations of IDA on the amplitude of CAPs. Reductions in the amplitude of the rat sciatic nerve by 3 different concentrations of IDA, measured 5 minutes after its application. The concentrations of IDA were 16 μM, 80 μM and 160 μM. The amplitude of the CAPs was unchanged by the administration of the IDA.

Figure 3 ; Distribution and concentration of ropivacaine in rat maxilla. After 0.5% 3H-ropivacaine was infiltrated into the right palatal mucosa proximal to the first molar without (○) or with (•) 10 μg/mL epinephrine, the radioactivity in (A) right palatal mucosa, (B) left palatal mucosa, (C) right maxilla part, or (D) left maxilla part was measured with the liquid scintillation counter. The concentration (ng/mg wet weight) of ropivacaine was calculated by the specific radioactivity. Data are mean ± SD (n = 7). *P < .05 versus the ropivacaine group.
Mikiko Yamashiro,
Shuichi Hashimoto,
Asako Yasuda, and
Katsuhisa Sunada
<bold>Figure 3</bold>
Figure 3

Distribution and concentration of ropivacaine in rat maxilla. After 0.5% 3H-ropivacaine was infiltrated into the right palatal mucosa proximal to the first molar without (○) or with (•) 10 μg/mL epinephrine, the radioactivity in (A) right palatal mucosa, (B) left palatal mucosa, (C) right maxilla part, or (D) left maxilla part was measured with the liquid scintillation counter. The concentration (ng/mg wet weight) of ropivacaine was calculated by the specific radioactivity. Data are mean ± SD (n = 7). *P < .05 versus the ropivacaine group.