Clin Exp Otorhinolaryngol.  2020 Nov;13(4):381-388. 10.21053/ceo.2019.01753.

Effects of Amniotic Membrane Extract on the Hyperplastic Response of the Middle Ear Mucosa in a Bacterially-Induced Otitis Media Rat Model: A Preliminary Study

• Park JH
• Kim HB
• Ko SH
• Kim BH
• Lim YS
• Park SW
• Song JJ
• Cho CG

• KMID: 2508514
• DOI: http://doi.org/10.21053/ceo.2019.01753

Abstract

Objectives
. Human amniotic membrane extract (AME) is known to contain numerous bioactive factors and anti-inflammatory substances. However, the anti-inflammatory effects of AME on the middle ear (ME) mucosa are unclear. This study assessed the effects of AME on the growth of the ME mucosa in response to bacterially-induced otitis media (OM).
Methods
. OM was induced by inoculating nontypeable Haemophilus influenzae (NTHi) into the ME cavity of rats. ME mucosal explants were cultured in AME concentrations of 0, 5, 10, or 50 μg/mL. The area of explant outgrowth was measured in culture and analyzed at 1, 3, 5, and 7 days after explantation. The expression of Ki-67, mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in the explants was also evaluated using quantitative polymerase chain reaction (PCR) and immunocytochemistry (ICC).
Results
. The NTHi-induced ME mucosa growth increased gradually over the 7-day culture period in all explants at different AME concentrations. There was a trend for mucosal growth inhibition at higher concentrations of AME, although the growth was not significantly different among the groups until day 5. The ME mucosal explants treated with the 50 μg/mL concentration of AME showed significantly suppressed growth on postexplantation day 7 compared with other explants on the same day. PCR and ICC staining revealed that the expression of Ki-67, MUC5AC, TNF-α, and IL-10 further decreased in the explants with higher concentrations of AME than in those with lower concentrations of AME.
Conclusion
. Our results showed that higher concentrations of AME reduced the mucosal proliferative response in bacterial OM in rats. These findings provide evidence that AME has an influence on the inflammatory and proliferative responses to NTHi infection in ME mucosa.

Keyword

Otitis Media; Amniotic Membrane; Haemophilus Influenzae; Rat

Figure

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  Fig. 1. Explant outgrowth evaluation. The diameter of explant outgrowth, which was approximately circular (blue dotted line), was measured, and its area was calculated in pixels using a microscopic computerized image analysis program (scale bar=100 μm).

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  Fig. 2. Outgrowth of infected middle ear mucosal explants cultured in different concentrations of amniotic membrane extract (AME). The growth of explants with 50 μg/mL AME was significantly inhibited on postexplantation day 7 compared to those with lower concentrations of AME (mean±standard error mean).

• 

  Fig. 3. Expression of Ki-67 (A), mucin 5AC (B), tumor necrosis factor-α (C), and interleukin-10 (D) in infected middle ear mucosa in different amniotic membrane extract concentrations at various time points after explantation.

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  Fig. 4. Immunocytochemical staining of Ki-67, mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in infected middle ear mucosa explants on postexplantation day 3. Immunocytochemistry shows decreased expression in the explants with higher concentrations of amniotic membrane extract (AME; ×400, scale bar=50 μm).

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