Neuropeptide immunofluorescence in human nasal mucosa: assessment of the technique for vasoactive intestinal peptide (VIP)

Abstract

Neuropeptides are important neurotransmitters in nasal physiology and the increasing knowledge of their role in nasal diseases brings new therapeutic perspectives. The investigation of human nasal mucosa neuropeptides is based mostly on immunocytochemistry, a complex approach whose resulting factors may be variable. Aiming to make this kind of research available, an immunofluorescence approach for vasoactive intestinal peptide (VIP) in human nasal mucosa is proposed and evaluated.

Study design: Transversal cohort.

Material and method: Human inferior turbinate samples were obtained at time of nasal surgery from eight patients. The samples were fixed in Zamboni solution (4% phosphate-buffered paraformaldehyde and 0.4% picric acid), snap-frozen and stored at -70 degrees C. 14 microm sections were then obtained. Immunofluorescence staining for VIP (Peninsula Laboratories) was performed and its images documented by conventional photography. The method's specificity, sensitivity and reproducibility of execution were evaluated. Additionally, the reproducibility of interpretation of results was evaluated through the comparison of staining scores (0 to 4) attributed to the images by six observers.

Results: The results showed the approach to be very specific and sensible, besides being reproducible in its execution. The interpretation of results may depend on the observer's accuracy in judging immunofluorescence images, but it showed uniformity.

Conclusion: The proposed method was highly useful for research purposes in neuropeptides in human nasal mucosa.

Figure 1

Example of labeling grade 1. Rare fibers identified (arrows). Artifact (A) and tissue auto-fluorescence (T).

Figure 2

Example of labeling grade 2. Sparse fibers (arrows). Tissue auto-fluorescence (T) and artifacts (A).

Figure 3

Example of labeling grade 3. Numerous fibers (arrows). Tissue auto-fluorescence (T) and artifacts (A).

Figure 4

Examples of labeling grade 4. Many fibers (arrows), and it is possible to follow their pathway intertwined with glandular structures. More marked tissue auto-fluorescence (T) and artifacts (A).

Graph 1

Distribution of grades (axis Y) attributed to photos (axis X) for each observer (color points).