The Histopathological Difference Between Oral Lichen Planus and Oral Lichenoid Mucositis in Neurogenic Inflammation

To explore the character of neurogenic inflammation in the inflammation of oral lichenoid reactions (OLR) and compare the difference between oral lichen planus (OLP) and oral lichenoid mucositis (OLM). Methods: The patient-based case control study applied twelve antibodies to check 74 biopsy specimens by the immunohistochemistry technique (IHC). Participants included 28 cases of oral lichen planus (OLP), 16 cases of contact stomatitis from dental restorative materials (OLM-dental) cases, 14 cases of mucosal reaction to systemic drug administration (OLM-drug), 15 cases of contact stomatitis from topical chemical exposure (OLM-contact) and one traumatic fibroma (TF) case. Twelve antibodies are anti-mast cell chymase (MCC), anti-calcitonin gene-related peptide (CGRP), anti-substance P (SP), anti-neuropeptide Y (NPY), anti-neurofilament heavy polypeptide (NF-H), anti-vasoactive intestinal peptide (VIP), anti-neurotrophin 3 (NT-3), anti-interferon gamma (INFG), anti-cytokine IL-22 (IL-22), anti-CD3 (CD3), anti-CD8 (CD8) and anti-CD4 (CD4). in the epithelial region. Mucosal reaction to systemic drug administration (OLM-drug) demonstrates a weaker expression of CD8 in epithelial region and INFG in the subepithelial region. polymer with DAB chromogen, but Dako envision polymer is applied for anti-mast cell chymase. Antibody dilution buffer was purchased in Abcam. MCC: anti-mast cell chymase; CGRP: anti-calcitonin gene-related peptide; SP: anti-substance P; NPY: anti-neuropeptide Y; NF-H: anti-neurofilament heavy polypeptide; VIP: anti-vasoactive intestinal peptide; NT-3: anti-neurotrophin 3; INFG: anti-interferon gamma; IL-22: anti-cytokine IL-22; CD3: anti-CD3; CD8: anti-CD8; CD4: anti-CD4.


Introduction
Oral lichenoid reaction (OLR) refers to oral lichen planus (OLP) or oral lichenoid mucositis (OLM). Oral lichenoid mucositis could be one of three conditions, which include lichenoid contact stomatitis from dental restorative materials (OLM-dental), mucosal reaction to systemic drug administration (OLM-drug), and contact stomatitis from topical chemical exposure (OLM-contact). These four oral mucosal conditions share a similar clinical appearance that is white reticular striations on erythematous mucosal base.
An incisional biopsy is required to confirm a definitive diagnosis there is no gold standard that can be followed [2] please see Figure 2.
The pathogenesis of OLP and OLM is categorized in T-cell mediated hypersensitivity type IV. However, the difference in subtypes between OLP and OLM is not clear. Neurogenic inflammation is a pathological process of a mutual interaction between neuropeptides and inflammatory cells, while it is proved in the experimental study level [3,4]. The purpose of this case-control study by using human tissue specimens is to detect different pathways between OLP and OLM. It will benefit for the histopathological diagnosis.

Materials and Methods
The case-control study used twelve antibodies to check biopsy specimens of 28 OLP cases and 45 OLM cases by immunohistochemistry (IHC) technique. OLM cases included 16 OLM-dental, 14 OLM-drug and 15 OLM-contact specimens. In addition, a traumatic fibroma (TF) case, was arranged in the study as a non-specific inflammation control. Participants aged between 28- Specimens in four study groups were numbered alphabetically only by a histologist (AK) herself. The result was unveiled after the scoring and before the data analysis. For avoiding bias, we have positive controls, negative controls, a non-specific inflammation control, the crucial inclusion and exclusion standard in each step. showed the similar feature as OLP, but has scattered eosinophils.

Inclusion, Exclusion and Preparation
OLM-contact showed the similar feature as OLP but has patched distribution please see Figure 2.  d.
e. OLM-dental (contact stomatitis from dental restorative materials): The similar feature as OLP, but has scattered plasma cells. e.
f. OLM-contact (contact stomatitis from topical chemical exposure): The similar feature as OLP, but has patched distribution.
The medical laboratory technologist (AK) sectioned specimens (4um thickness, 3-5mm length and 2-3mm wideness) from paraffin tissue blocks, which were stored in the pathology archive of the OBS. She mounted 5 -8 specimens on one glass slide. Therefore, 88 specimens were arranged in 14 slides. We named it "Minor Array Plate". Totally 252 slides (14 x 18) were prepared for IHC study. Exclusion is uneven staining, unusual weak or absent staining, background or artifactual staining and inadequate amount of a specimen.

Antibodies and Optimal Dilution
Twelve primary antibodies and the antibody dilution buffer were purchased from Abcam (Toronto, ON, Canada M5W 0E9).
Second antibody (mouse IgG) and reagents were bought from Inter Medico (Markham, ON, Canada L3R 6E9). All antibodies are able of reacting to human tissue. We toned the optimal antibiotic dilution in positive control specimens that were recommended by Abcam and published reference. Negative controls were obtained Volume 27-Issue 4 by replacing the primary antibody with mouse IgG. Please see details in Table 1. Note: All primary antibodies react to human tissue. The scoring principle is score 1: < 10% and/or mild stain; score 2: 10-50% and/or mild-moderate stain; score 3: 50-90% and/or moderate-strong stain; score 4: >90% and/or strong stain. The scoring procedure was conducted by two researchers (EL and TB) and an oral pathologist (YG) respectively for 18 times. The scoring protocol is to score the staining outcome to epithelial region, subepithelial region and submucosal region separately. Each case was marked by three scores. We called it "Sandwich Scoring". Please see details in Traumatic fibroma showed a nest-like staining pattern. Those were recorded in the scoring data as well please see details in table 2.   I. Scoring principle: Score 1: < 10% and/or mild stain; Score 2: 10-50% and/or mild-moderate stain; Score 3: 50-90% and/ or moderate-strong stain; Score 4: >90% and/or strong stain.

Analysis of the Data
The study size are 28 cases in OLP group, 16 cases in OLMdental group, 14 cases in OLM-drug group and 15 cases in OLMcontact group. The outcome of immunopositive scores in each group was obtained by the mean value of all cases. It is surprised that the standard deviation for the mean value in each group is one.
It means specimens in each study group showed the same specific immunopositive pattern. Therefore, we did the comparison and contrast based on their patterns, rather than on their individual data. We call it "Pattern Comparison". The comparing baseline is represented by the non-specific inflammation control. There is a remarkable difference if the mean score in a study group is higher than the score in the same region in the non-specific inflammation control please see details in table 2.  We received all immunopositive outcomes in four study groups (Please see Table 2 and

Vasoactive intestinal peptide (VIP) and Neurotrophin 3 (NT-3) [4].
We received five immunopositive outcomes in four study groups (Please see Table 2 and Figure 5). VIP and NT-3 showed negative results in all groups. Traumatic fibroma (TF) was used as a baseline control for the comparison. There is a significantly stronger expression of CGRP, SP, NPY and NF-H in the subepithelial region in four study groups, while the stronger expression of MCC in the subepithelial region was only found in OLM-dental and OLM-drug groups. The remarkable increase of CGRP in epithelial, subepithelial and submucosal regions was noticed in four study groups, but the strongest one is OLM-dental group. The significant decrease of NPY in epithelial region was identified in OLP, OLM-dental and OLMdrug groups. The only notable decrease of NF-H staining in the epithelial region was in the OLP group. The intensity of neurogenic inflammation is organized in a descending order of OLM-dental group, OLM-contact group, OLM-drug group and OLP group. INFG is produced by inflammatory cells and mucosal epithelium.
There was no difference between traumatic fibroma and four study groups in the epithelial regions. In addition, IFNG promotes the Th1 differentiation first, and then Th1 produces INFG consequently

1.
Neurogenic inflammation does play a role in the inflammatory process of oral lichenoid reaction (OLR), but different types of OLR have different levels of involvement.

2.
Lichenoid contact stomatitis from dental restorative materials (OLM-dental) significantly involves in neurogenic inflammation. The stronger expression of CGRP in epithelial, subepithelial and submucosal regions is special.

3.
Oral lichen planus (OLP) most likely involve in cytolysistype of T-cell mediated hypersensitivity type IV. The partial loss of Neurofilament and Neuropeptide Y in the epithelium is unique.

4.
Mucosal reaction to systemic drug administration (OLMdrug) most likely involve in delayed-type of T-cell mediated hypersensitivity type IV. The weaker expression of CD8 in epithelial region and INFG in the subepithelial region is distinctive.

Conflicts of Interest
All authors declare they have no conflict of interest.

Ethical Approval
All procedures performed in this study were in accordance with the principles of the