A comprehensive analysis of the novel tubarial glands

Methods

Summary

To meet our overarching objective, this study proposes to use 14 male and 14 female, clinical grade, lightly embalmed cadavers. Major SGs, minor SGs, and TGs will be dissected from the cadavers for histological analysis using immunohistochemistry (IHC) with the PSMA-ligands, and hematoxylin and eosin (H&E) staining with the PAS mucin stains. The proposed histological measures include serous and mucus acini density and volume, serous to mucus cell ratio, mucin index, and uptake of PSMA ligands for each sample. Comparisons will be made between the TGs and the SGs, as well as between male and female TGs.

Challenges

A potential limitation is associated with the embalming process. With light embalming there is the possibility of artifacts, which are artificial structures or tissue alterations. These structures and alterations can result in misinterpretations and difficulties with analysis due to the tissue structures differing from how they were prior to embalming. For example, light embalming can result in loss of integrity of the intra- and extracellular compartments of the cells due to inadequate protein cross-linking caused by the formalin. To mitigate the effect of artifacts, biopsy samples of the glands will be received from the University of Calgary’s Department of Pathology. These pathological samples will not have undergone embalming and will be able to serve as a reference to compare our lightly embalmed samples and clarify if any artifacts are present. 

Pre Analysis Plan

Research Question 1: What are the histological similarities and differences between the TGs and the major and minor SGs?

1.1 Hypothesis: We hypothesize that the histological profile of the TGs is more congruent with the minor SGs than the major SGs.

1.2 Expectations: We believe that the TGs will more closely resemble minor SGs than major SGs. Minor SGs contain the “flat submucosal glandular structures” that were used to describe the TGs. Additionally, in the images provided by Valstar et al., the TGs’ acini arrangement more closely resembled that of the minor SGs compared to the major SGs.

1.3 Rationale: Due to the TGs not producing amylase, Valstar et al.’s hypothesized role for the TGs in salivation is contested and therefore more research into their histology is required to understand what their role could possibly be. Additionally, Valstar et al. noted that the TGs bear similarities to the minor SGs, without any further discussion as to what those similarities were, prompting the need for additional research.

Research Question 2: Are there sex-based differences in the TGs between males and females?

2.1 Hypothesis: We hypothesize that no differences exist between the male and female TGs other than size.

2.2 Expectations: We expect the female TGs to exhibit a smaller average cranio-caudal length compared to the male TGs, as differences in size have been described in previous SG studies. However, we do not expect there to be other differences between the TGs of males and females because previous studies on the SGs have demonstrated that SGs are relatively homogenous between the sexes in terms of structural and histological measures.

2.3 Rationale: As discussed in the Sex and Gender Considerations section, there are some known differences between male and female SGs and in salivation rates, which provide justification for questioning if there are any differences between the male and female TGs.

Tissue sample analysis: After the IHC and H&E staining processes are complete, comparisons between the TGs and major and minor SGs, as well as between male and female TGs will be made. Specifically, comparisons will be made regarding the serous and mucus acini density and volume, serous to mucus cells ratio, mucin index, and uptake of PSMA-ligands. Samples will be analyzed using a standard light microscope. Microscopy images will be analyzed using trainable Weka Segmentation machine learning software on ImageJ as well as area and density tools on ImageJ. Standard descriptive statistics and two-tailed unpaired t-tests will be used to analyze the results. A p-value of 0.05 will be considered significant.