Study design and patients
This was a retrospective study. We collected observation data based on the inclusion criteria; patients underwent microneurosurgery of the lingual nerve in Wakayama Medical University Hospital between July 2004 and December 2016 for lingual nerve injury caused by third molar extraction. Patients were classified into two groups: those who underwent microneurosurgery within 6 months after lingual nerve injury (early cases) and those more than 6 months after injury (later cases). This was in accordance with previous study of lingual nerve recovery , because some patients with lingual nerve injury can spontaneously recover the sensory and taste function within 3 months. Collection of observational data was based on opt-out consent, and collection of traumatic neuromas and normal lingual nerves was based on written informed consent. This study was performed in accordance with the Declaration of Helsinki for medical protocols and was approved by the Wakayama Medical University Institutional Review Board (Nos. 1689 and 1698).
Evaluation of lingual nerve repair
The sensory and taste tests in the tongue were performed according to the procedures described by Fujita et al. . Briefly, the criteria for achieving FSR were static 2PD < 20 mm and the presence of superficial pain/tactile sensation, light touch, and brush-stroke direction without overreaction. The criterion for functional taste recovery was improving at least one kind of taste perception compared with the results of the preoperative in taste test using Taste Disc (Sanwa Kagaku Kenkyusyo Co., Nagoya, Japan; salty: sodium chloride 1.0 mol/L, sweet: sucrose 1.0 mol/L, sour: acetic acid 0.4 mol/L, and bitter: quinine 0.1 mol/L).
All patients were seen and evaluated by one of the authors (S F). The microneurosurgery to repair the injured lingual nerve was performed by one (S F) of the authors, when patients met the criteria for performing the microneurosurgery. The criteria were as follows: (1) no signs of recovery during close follow-up for at least 3 months (2) good in general medical condition, (3) two-point discrimination (2PD) > 20 mm in the affected area, (3) no sensation observed against cold (0 °C) or hot water (42 °C) during a temperature test in the affected area, (4) no sensation against salt, sweet, sour, or bitter observed during a taste test in the affected area, (5) no sensation against sharp touch observed during a pin-prick test in the affected area, (6) no sensation against any directions observed during a brush-stroke test in the affected area, and (7) a difference in the Semmes-Weinstein monofilament test between the affected and non-affected side. Microneurosurgery procedures for lingual nerve injury were performed in all cases as previously reported . Briefly, the lingual nerve was approached by an intraoral mucosal incision and lingual flap reflection, and the scar tissues around the injured site were removed. The traumatic neuroma was excised, and direct end-to-end epineural nerve sutures without tension were performed at eight or more sites around the stump using 8-0 or 9-0 nylon.
Immunohistochemistry in traumatic neuroma samples
The excised traumatic neuroma samples in the microneurosurgery were collected from 2013 onward and fixed in 10% neutral buffered formalin overnight. Thirty traumatic neuroma samples were embedded in paraffin and sliced longitudinally. Endogenous peroxidase activities were removed by incubation with 0.3% hydrogen peroxide for 1 h. Non-specific immunoreactivities were blocked by incubation with Blocking One (Nacalai Tesque, Inc., Kyoto, Japan) for 1 h. The sections were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies conjugated with peroxidase for 1 h. The dilution ratio of the anti-S100 beta antibody was 1/1000. Secondary antibodies were detected using 3, 3′-diaminobenzidine solution with 0.01% hydrogen peroxide, and methyl green was used for nuclear staining.
Quantification of Schwann cells in traumatic neuroma samples
Images of the tissue sections were acquired with a microscope (Eclipse E600, Nikon, Japan), and a section from approximately the middle of each specimen was selected for analysis. To determine the abundance ratio of Schwann cells, the area of immunoreactivity against anti-S100β antibodies was measured using ImageJ software in three fields of view from the central side of the traumatic neuroma to the peripheral side. The abundance ratio of Schwann cells in each sample was calculated as the average of the immunoreacted area against anti-S100β antibodies per tissue area in three fields of view [23,24,25].
The primary outcome was the ratio difference of functional sensory or taste recovery at 12 months after microneurosurgery between early and later cases. Secondary outcomes were ratio difference of allodynia appearance at 12 months after microneurosurgery between early and later cases, ratio difference of sensory or taste function recovery at 24 months after microneurosurgery between early and later cases, differences between early and later cases of ratio of functional sensory or taste recovery within 12 months after the microneurosurgery compared with those 12 to 24 months after microneurosurgery, and the abundance ratio of Schwann cells in traumatic neuroma samples between early and later cases.
All data were statistically analyzed using JMP Pro 12 (SAS Institute Inc., NC, USA). Statistical comparisons were performed between early and later cases for FSR, functional taste recovery, and the presence of allodynia using Fisher’s exact test. The distance in 2PD test and abundance ratio of Schwann cells were statistically compared between early and later cases using Student’s t tests. For all analyses, P < 0.05 was considered to be statistically significant.