## 3 Results and Discussion
### Histological Evaluation
#### 3.6.1 Olfactory Nasal Epithelial Tissue  
The microscopic images of the olfactory nasal epithelial tissues before and after exposure to the different microparticle formulations are depicted in Figure 5. The tissues were stained with haematoxylin (blue-purple stain of the nuclei), aqueous eosin Y (pink stain of the cytoplasm and extracellular matrix), and Alcian blue (bright blue stain of the mucus).  
The micrographs of the olfactory sheep nasal tissue in the intact control group (Figure 5A, which is freshly dissected tissue before it was mounted in the diffusion chamber) showed that a layer of mucus (i.e., bright blue colour) was present on the surface of the epithelium, while the pseudostratified ciliated columnar epithelial cells appeared intact with goblet cells interspaced between them. [PERSON]'s glands are visible in the tissue below the epithelial layer (illustrated by the concentrated bright blue stain in the lamina propria) [24]. When comparing the mucus layer and the presence of goblet cells and [PERSON]'s glands in Figure 5A (untreated control) and Figure 5B (insulin-treated control), it is apparent that no physical damage occurred in the olfactory epithelial tissue during exposure to insulin alone (control group) in the permeation experiment.  
In a previous study, it was shown that _A. vera_ gel and whole leaf materials in solution caused some loosening and detaching of epithelial cells from excised sheep nasal epithelial  
Figure 5: Micrographs of olfactory sheep nasal epithelial tissues of (**A**) the intact control, (**B**) after exposure to insulin in the control group, (**C**) after exposure to microparticles consisting of Eudragit® L100 and _A. vera_ polysaccharides prepared by the emulsion-solvent evaporation, (**D**) after exposure to microparticles consisting of _A. vera_ polysaccharide prepared by the emulsion-solvent evaporation, and (**E**) after exposure to microparticles consisting of _A. vera_ polysaccharide prepared by ionic gelation.  
tissues [24]. In this study, some physical damage can be observed on the olfactory sheep nasal tissues after exposure to the different microparticle formulations, albeit to a smaller degree, as reported by [PERSON] et al. [24]. In Figure 5C, some mucus, goblet cells, and Bowman's glands are observed, which are indicators of an intact epithelial layer, similar to the untreated and insulin-treated control groups (Figure 5A,B). The tissue treated with microparticles consisting of _A. vera_ polysaccharide prepared by the emulsion-solvent evaporation (Figure 5D) showed the epithelial layer is still intact, although there are some signs of detachment between the epithelial layer and the lamina propria, indicating some damage may have started to occur after 2 h treatment. Lastly, the tissue treated with the microparticles consisting of _A. vera_ polysaccharides prepared by ionic gelation (Figure 5E) showed relatively more damage than the other microparticles (Figure 5C,D). The epithelial layer appears disintegrated, and the lamina propria showed signs of delamination. This indicated some protective activity of the Eudragit\({}^{\text{\textregistered}}\) L100 incorporated within the microparticles prepared by emulsion-solvent evaporation.