In vivo hamster cheek pouch subepithelial ablation, biomaterial injection, and localization: pilot study

Abstract

Significance: The creation of subepithelial voids within scarred vocal folds via ultrafast laser ablation may help in localization of injectable biomaterials toward a clinically viable therapy for vocal fold scarring.

Aim: We aim to prove that subepithelial voids can be created in a live animal model and that the ablation process does not engender additional scar formation. We demonstrate localization and long-term retention of an injectable biomaterial within subepithelial voids.

Approach: A benchtop nonlinear microscope was used to create subepithelial voids within healthy and scarred cheek pouches of four Syrian hamsters. A model biomaterial, polyethylene glycol tagged with rhodamine dye, was then injected into these voids using a custom injection setup. Follow-up imaging studies at 1- and 2-week time points were performed using the same benchtop nonlinear microscope. Subsequent histology assessed void morphology and biomaterial retention.

Results: Focused ultrashort pulses can be used to create large subepithelial voids in vivo. Our analysis suggests that the ablation process does not introduce any scar formation. Moreover, these studies indicate localization, and, more importantly, long-term retention of the model biomaterial injected into these voids. Both nonlinear microscopy and histological examination indicate the presence of biomaterial-filled voids in healthy and scarred cheek pouches 2 weeks postoperation.

Conclusions: We successfully demonstrated subepithelial void formation, biomaterial injection, and biomaterial retention in a live animal model. This pilot study is an important step toward clinical acceptance of a new type of therapy for vocal fold scarring. Future long-term studies on large animals will utilize a miniaturized surgical probe to further assess the clinical viability of such a therapy.

Keywords: ablation of tissue; hamster cheek pouch; in vivo; nonlinear microscopy; ultrafast lasers; vocal fold scarring.

Fig. 1

In vivo setup for ablation and biomaterial injection in hamster cheek pouch. (a) Hamster cheek pouch everted and clamped with a Desmarres Chalazion clamp. (b) A zoomed-in image showing the black tattoo ink marks, some blood vessels, and the treated area that underwent ablation and is now ready for the biomaterial injection. (c) A 30-gauge needle piercing through the epithelium and aimed toward the ablated void. (d) The rhodamine-tagged PEG30 appears as a red-orange oval under the epithelium, localized inside the void. (e) The same FOV as in (d) with the FITC filter showing the fluorescence of the rhodamine with blood stains appearing as dark red.

Fig. 2

Nonlinear imaging of hamster cheek pouches after surgery and rhodamine-tagged PEG30 injection. (a) A cross-sectional image and (b) a 3D image of the biomaterial-filled void 2 weeks postoperatively in the nonscar group. Rhodamine particles (red) were resolved using TPF imaging and are localized within the subepithelial void. Collagen fibers (green) were resolved using SHG imaging. Dashed lines in (a) show the assumed void margins. The length of the void extends outside the FOV of this figure. The top $25\mu \mathrm{m}$ of epithelium was removed in (b) to clearly visualize rhodamine within the subepithelial void. (c) SHG image of collagen fibers in the nonscar group in a region adjacent to the biomaterial-filled void shown in (a) and (b). Fibers appear highly disorganized in (c), as expected for healthy tissue. Collagen fiber structure near the void margins in (a) resembles those shown in (c), confirming that the ablation process does not cause scarring. (d) A cross-sectional image and (e) a 3D image of the biomaterial-filled void 2 weeks postoperatively in the scar group. (f) SHG image of collagen fibers in the scar group. Fiber alignment/organization in (d) indicates presence of scar tissue.

Fig. 3

Histology of nonscarred and scarred tissues 2-weeks and 1-week after surgery, respectively. (a) Collagen I antibody staining reveals the void created in the nonscar group 2-weeks postsurgery with (b) H&E staining showing the rhodamine particles. (a) and (b) Consecutive slides ($4\mu \mathrm{m}$ separation). (c) A scarred tissue shows a collapsed void under the epithelium. The dashed oval is the needle entry location. (d) A slide $\sim 28\mu \mathrm{m}$ away from the slide shown in (c) shows complete separation from the underlying tissue, which may have been caused by fixation and sectioning. The dashed square indicates the FOV shown in (e). (e) A zoomed-in image of the slide shown in (d). Rhodamine particles are seen in the separated tissue layer. Black and green arrows in (a)–(e) indicate void margins and rhodamine particles, respectively.