Regional Differences following Partial Salivary Gland Resection

Abstract

Regenerative medicine aims to repair, replace, or restore function to tissues damaged by aging, disease, or injury. Partial organ resection is not only a common clinical approach in cancer therapy but also an experimental injury model used to examine mechanisms of regeneration and repair in organs. We performed a partial resection, or partial sialoadenectomy, in the female murine submandibular salivary gland (SMG) to establish a model for investigation of repair mechanisms in salivary glands (SGs). After partial sialoadenectomy, we performed whole-gland measurements over a period of 56 d and found that the gland increased slightly in size. We used microarray analysis and immunohistochemistry (IHC) to examine messenger RNA and protein changes in glands over time. Microarray analysis identified dynamic changes in the transcriptome 3 d after injury that were largely resolved by day 14. At the 3-d time point, we detected gene signatures for cell cycle regulation, inflammatory/repair response, and extracellular matrix (ECM) remodeling in the partially resected glands. Using quantitative IHC, we identified a transient proliferative response throughout the gland. Both secretory epithelial and stromal cells expressed Ki67 that was detectable at day 3 and largely resolved by day 14. IHC also revealed that while most of the gland underwent a wound-healing response that resolved by day 14, a small region of the gland showed an aberrant sustained fibrotic response characterized by increased levels of ECM deposition, sustained Ki67 levels in stromal cells, and a persistent M2 macrophage response through day 56. The partial submandibular salivary gland resection model provides an opportunity to examine a normal healing response and an aberrant fibrotic response within the same gland to uncover mechanisms that prevent wound healing and regeneration in mammals. Understanding regional differences in the wound-healing responses may ultimately affect regenerative therapies for patients.

Keywords: extracellular matrix; fibrosis; macrophages; regeneration; submandibular gland; wound repair.

Figure 1.

Response of female adult submandibular salivary glands to partial gland resection. (A) Image of partially resected female adult submandibular salivary gland (top left) and excised gland piece (bottom left) compared to unmanipulated control gland (right) at day 0. Image shows proximal to distal gland orientation with the sublingual gland (SLG) indicated with black dotted line. (B) Length and width of the excised gland pieces were measured with calipers in millimeters. Length (n = 32) and width (n = 32). (C) Weight of the excised gland piece was obtained in milligrams (n = 32). (D) Gland length was measured in situ with calipers after partial resection with immediate tissue harvest (day 0) (n = 9), 3 d postresection (day 3) (n = 11), 14 d postresection (day 14) (n = 8), and 56 d post resection (day 56) (n = 4) and normalized to gland length removed at the time of resection. (E) Quantification of gland widths measured with calipers and normalized to width of the excised gland piece. (F) Quantification of gland weight normalized to excised gland weight measured at all time points. Error bars are SEM. Statistical tests: 1-way analysis of variance Tukey’s honestly significant difference, *P ≤ 0.05.

Figure 2.

Differential gene expression in partially resected glands at day 3 and day 14. Total RNA was isolated from adult female submandibular salivary glands that were subjected to mock surgery and harvested at day 3 (control C, blue), partial resection and harvested at day 3 (day 3, red), and partial resection and harvested at day 14 (day 14, purple). n = 3 animals for each treatment. Transcriptomes were obtained following hybridization with Clariom S arrays and analyzed with TAC software. (A) Principal component analysis (PCA) was performed to examine the variance between the samples. (B) The total genes were quantified that were changed >2-fold relative to C and having a 1-way between-subject P < 0.05 corrected for false-discovery rate (FDR) using the Benjamini-Hochberg procedure. (C, D) Volcano plot for differentially expressed genes at day 3 (C) and day 14 (D) relative to C with genes increased greater than 2-fold in red and decreased greater than 2-fold in blue. (E) Enriched Reactome (R-MMU) and Gene Ontology (GO) categories at each time point were identified with Metascape.

Figure 3.

Cell cycle entry is transiently increased globally in both secretory epithelial and stromal cells after partial salivary gland resection. (A) Immunohistochemistry (IHC) was performed in multiple tissue sections representing all regions of the gland to detect the proliferation marker, Ki67 (white), relative to 4′,6-diamidino-2-phenylindole (DAPI) (blue) in control female submandibular glands day 0 or 3, 14, or 56 d after partial resection. (B) Quantification of Ki67⁺ cells normalized to total DAPI⁺ cells. Day 0 (n = 3 glands), day 3 (n = 3), day 14 (n = 4), and day 56 (n = 4). Statistical test: 1-way analysis of variance Tukey’s honestly significant difference **P ≤ 0.01. (C) IHC to detect proliferating epithelium. EpCAM⁺ (red) and Ki67⁺ (white) at day 0 and day 3. (D) IHC to detect proliferating Aqp⁺ acinar cells with Aqp5 (green) and Ki67 (white). (E) Diagram showing periductal area (I, purple), acini (A, gray), ducts (D, green), and vasculature (V, red). (F) IHC to detect proliferating fibroblasts with vimentin (VIM, purple) and Ki67 (white) at day 0 and day 3. Most of the VIM⁺/Ki67⁺ cells are in the periductal regions. (G) Quantification of Ki67⁺/EpCam⁺, Ki67⁺/Aqp5⁺, or Ki67⁺/Vim⁺ cells at day 0 and day 3, normalized to DAPI in images from 3 glands with 3 to 4 images from each gland. Statistical test: Student’s 2-tailed t test, *P ≤ 0.05, **P ≤ 0.01. (H) IHC for Aqp5 (green) at day 0 or 3, 14, or 56 d after partial resection. (I) Quantification of Aqp5⁺ area normalized to DAPI. (J) IHC for the mucin functional marker, Muc10 (green), at day 0 or 3, 14, or 56 d after partial resection. (K) Quantification of Muc10⁺ area. IHC for Muc10 and Aqp5 was obtained from multiple regions in the gland, day 0 (n = 4 glands), day 3 (n = 3), day 14 (n = 4), and day 56 (n = 4). Error bars are SEM. Scale bars, 100 microns.

Figure 4.

An aberrantly proliferative region of the gland shows reduced secretory capacity following partial gland resection. (A) Cartoon depicting the general location of the aberrant repair response (black dotted line) with respect to the sublingual gland (SLG), submandibular gland (SMG), and resection line (red dotted line). (B) Hematoxylin and eosin (H&E) staining within a region of normal morphology (global area) at day 14 after resection. (C) H&E staining within a region of aberrant morphology, with a ductal-rich pattern (indicated by arrowheads) at day 14 after resection (local area). (D) Immunohistochemistry (IHC) to detect Ki67⁺ cells (white) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) in aberrant local area at 0, 3, 14, and 56 d. (E) Quantification of Ki67⁺ cells in the local region relative to global region at all time points. (F) IHC for Aqp5⁺ (red) and DAPI (blue) in local aberrant region. (G) Quantification of Aqp5⁺ area in the local and global regions. (H) IHC for Muc10⁺ cells (green) and DAPI (blue) in local region. (I) Quantification of Muc10⁺ area normalized to DAPI in local versus global region. (J) Vimentin (VIM) (purple) and Ki67 by IHC. (K) Quantification of VIM⁺/Ki67⁺ cells at all time points in the local region compared to the global. Statistical analysis: Student’s 2-tailed t test, *P ≤ 0.05, **P ≤ 0.01. Global measurements from representative images: day 3, n = 3 glands; day 14, n = 4; and day 56, n = 4. Local measurements from representative images for Ki67: days 3 and 14 (n = 4) and day 56 (n = 3). Error bars, SEM. Scale bars, 100 microns.

Figure 5.

Partial gland resection transiently increases macrophages and extracellular matrix (ECM) deposition 3 d after resection that are sustained in localized aberrant regions. (A) Immunohistochemistry (IHC) for the macrophage marker, F4/80 (green), with 4′,6-diamidino-2-phenylindole (DAPI) (blue) at days 0, 3, 14, and 56. (B) Quantification of F4/80⁺ area normalized to DAPI. Statistical analysis: 1-way analysis of variance Tukey’s honestly significant difference, **P ≤ 0.01. (C) Overview image showing the localized and global areas with DAPI and F4/80; yellow dotted box shows 20× images depicted in D, scale bar 250 microns. (D) F4/80⁺ IHC and DAPI in the local aberrant areas at 3, 14, and 56 d. (E) Quantification of F4/80⁺ area in the local aberrant regions relative to the global. (F) IHC for the M2 macrophage marker, CD206 (magenta), F4/80 (green), with DAPI (blue) at days 0, 3, 14, and 56. (G) Quantification of CD206⁺ and F4/80⁺ colocalized area normalized to F4/80⁺ area. (H) Overview image showing the localized and global areas with CD206, F4/80, and DAPI; yellow dotted box shows 20× images depicted in I, scale bar 250 microns. (I) CD206⁺ and F4/80⁺ IHC with DAPI (blue) in the local aberrant areas at 3, 14, and 56 d. (J) Quantification of CD206⁺ and F4/80⁺ (gray) copositive area normalized to the local aberrant regions relative to the global F4/80⁺ area. (K) Masson’s trichrome staining at days 0, 3, 14, and 56. (L) Quantification blue area in Global Masson’s trichrome staining. (M) Overview image showing the localized and global areas with Masson’s trichrome; yellow dotted box shows 10× images depicted in N, scale bar 500 microns. (N) Masson’s trichrome stain in local aberrant regions at days 3, 14, and 56. (O) Quantification of Masson’s trichrome stain in the aberrant local regions. Statistical analysis: Student’s 2-tailed t test, *P ≤ 0.05, **P ≤ 0.01. All global measurements were taken from multiple, representative images (n = 3 for days 0 and 3, n = 4 for days 14 and 56). Images for local analysis were taken from multiple, representative images (n = 3). Error bars, SEM. Scale bars, 100 microns.