Fig 1.
Armamentarium for transcutaneous IG injection procedure.
In a clean, disinfected hood, the following armamentarium were prepared: A. Inhalation anaesthetic unit; B. Isoflurane-USP; C. 10% Povidine-iodine solution; D. Lubricant ophthalmic ointment; E. 70% Ethanol swabs; F. Masking tapes; G. Surgical scissors, stainless steel; H. Anatomical forceps, fine, stainless steel; I. Electric razor; J. Disinfected plastic stencil; K. Disinfected ruler; L. Paper ruler; M. Fine tip markers; N. 28G, 12.7 mm, 0.5 ml, U-100 insulin syringe; O. Working platform; P. Heated recovery pad.
Fig 2.
Anatomical Landmark Identification.
A. The cervical area of a sedated and restrained mouse was shaved and prepared for the procedure; B. Mark the midline from the lower lip (point A) to point B at the xiphoid process of the sternum (length of 19 mm) was traced, point C was marked 9 mm inferiorly to point A; C. The sites of IG injections (points D) were measured 2 mm laterally on each side at point C; D. A plastic stencil was created as a guide for injections, using the above landmarks and measurements.
Fig 3.
Intraglandular Injection Technique.
The syringe loaded with the solution was placed at one IG injection site, perpendicular to the mouse (A. Bird’s eye view; B. Lateral view).
Table 1.
Measurements as plotted on C57BL/6 and NOD mice.
Fig 4.
Further Validation of the Location of Submandibular Glands using an MRI Image and Plastic Stencil.
A. Sedated and restrained mouse with plotted landmarks (Points A, B, C, and D) for the non-surgical transcutaneous IG injections; B. T2-weighted MRI image of the mouse SMG. C. superimposed image of our landmarks and the frontal cross-sectional T2-weighted MRI image of the mouse SMG; D. A plastic stencil prepared with the same landmarks. E. Image of the exposed SMGs with the marked stencil placed.
Fig 5.
Submandibular Salivary Gland Localization and Injection in NOD Mice.
A. The cervical area of a sedated and restrained mouse was shaved and prepared for the procedure; B. Mark the midline from the lower lip (point A) to point B xiphoid process of the sternum (length of 18 mm) and point C was traced, 8 mm inferiorly to point A; C. The sites of IG injections (points D) were measured 2 mm laterally on each side at point B; D A plastic stencil was created as a guide for injections, using the above landmarks and measurements; E, F. The syringe loaded with the solution was placed at one IG injection site, perpendicular to the mouse (E. Bird’s eye view; F. Lateral view).
Fig 6.
Verification of salivary gland location using Trypan Blue Dye.
A. Exposed SMGs of a euthanized mouse after non-surgical, transcutaneous IG Trypan Blue injections; B. Harvested SMGs revealed uptake of the Trypan Blue dye; C. SMG area after excision of the glands, showing no spread of dye to the surrounding structures.
Fig 7.
Intraglandular injection of GFP + bone marrow mesenchymal stem cells in submandibular glands.
A-C; Confocal microscopy images demonstrating GFP + ve BM-MSCs in culture (5x, 10x, 20x; scale bar = 100 pixel = 26400.58 micron). D; Eppendorf tube with GFP + ve BM-MSCs emitting green fluorescence under ultra-violet lamp. E; Control Eppendorf tube with normal saline emitting no colour under ultra-violet lamp. F; Control Eppendorf tube with non-labelled MSCs emitting no colour under ultra-violet lamp.
Fig 8.
Histopathological Analysis of injected GFP + BM-MSCs in submandibular glands.
H&E sections demonstrate normal glandular architecture, whereas the unstained sections show green fluorescence due to GFP + ve MSC injections. Specimens were harvested on Day 1(A), Day 3 (B), and Day 7 (C) post IG injection. The green fluorescence is noticeably fading over time. The third row of images show the same sections with immunofluorescent staining of salivary epithelial cells (Pan CK, red; DAPI, blue) with the GFP+ MSC’s present.