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Abstract
Objective
Sensorineural hearing loss leads to the progressive degeneration of spiral ganglion cells (SGC). Next to postoperative fibrous tissue growth, which should be suppressed to assure a close nerve–electrode interaction, the density of healthy SGC is one factor that influences the efficiency of cochlear implants (CI), the choice of treatment for affected patients. Rolipram, a phosphodiesterase-4 inhibitor, has proven neuroprotective and anti-inflammatory effects and might also reduce SGC degeneration and fibrosis, but it has to pass the cellular membrane to be biologically active.
Methods
Lipidic nanocapsules (LNC) can be used as biodegradable drug carriers to increase the efficacy of conventional application methods. We examined the biological effects of rolipram and LNC’s core encapsulated rolipram on SGC and dendritic cell (DC) tumor necrosis factor-α (TNF-α) production in vitro and on SGC survival in systemically-deafened guinea pigs in vivo.
Results
Our results prove that rolipram does not have a beneficial effect on cultured SGC. Incorporation of rolipram in LNC increased the survival of SGC significantly. In the DC study, rolipram significantly inhibited TNF-α in a dose-dependent manner. The rolipram-loaded LNC provided a significant cytokine inhibition as well. In vivo data do not confirm the in vitro results.
Conclusion
By transporting rolipram into the SGC cytoplasm, LNC enabled the neuroprotective effect of rolipram in vitro, but not in vivo. This might be due to dilution of test substances by perilymph or an inadequate release of rolipram based on differing in vivo and in vitro conditions. Nevertheless, based on in vitro results, proving a significantly increased neuronal survival when using LNC-rolipram compared to pure rolipram and pure LNC application, we believe that the combination of rolipram and LNC can potentially reduce neuronal degeneration and fibrosis after CI implantation. We conclude that rolipram is a promising drug that can be used in inner ear therapy and that LNC have potential as an inner ear drug-delivery system. Further experiments with modified conditions might reveal in vivo biological effects.
Acknowledgments
This study was supported by the European Community 6th Framework Programme on Research, Technological Development and Demonstration (Nanotechnology-based Drug Delivery. Contract Number: NMP4-CT-2006-026556, Project acronym: NANOEAR). We would like to thank Dr Henning Voigt, Hannover Medical School, for his support in quality management.
Disclosure
The authors report no conflict of interest in this work.
Supplementary material
Vitality test
To determine the vitality of dendritic cells at the end of the experiment (day 10), a CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) was performed. In addition, the cells were subjected to microscopic inspection to note any morphological differences. This examination allows a trend statement about a possible influence of the test substances. The results of the viability test reveal possible cellular impairments caused by the test substances. Thus, a lower production of tumor necrosis factor-α (TNF-α) depends not only on the inhibition of cytokine production but also on an increased degeneration of cells. This assay is based on nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide (NADH), which catalyzes the conversion of the yellow methyltetrazolium to a violet formazan product. NADPH/NADH are only found in living cells. Consequently, a color change by 490 nm measured by photometric analysis is an indication of cell vitality. The resulting amount of formazan is directly proportional to the number of viable cells. Supplementary Table 1 shows the measured values.
AABR measurements
Acoustically evoked auditory brainstem response measurements were performed to determine the hearing threshold of all experimental animals before and after deafening for verification of the procedure’s success. Because the used system allows stimulation with a maximum of 90 dB SPL and all animals’ hearing thresholds reached or exceeded that level 7 and 21 days after deafening, the hearing thresholds are plotted at 90 dB (Supplementary Figure 1).
Figure S1 By frequency-specific AABR measurements on day 0, a normal hearing threshold was detected in all animals (blue and pink line; mean ± SD).
Notes: The results of the measurements on day 7 and day 21 are plotted as a red line. None of the animals had residual hearing after the deafening procedure. The hearing threshold of all animals was 90 dB SPL or above on day 7 and did not recover until the final measurement on day 21.
Abbreviations: AABR, acoustically evoked auditory brainstem response; SD, standard deviation.
Table S1 Vitality values of dendritic cells after incubation with the test compounds and LPS stimulation; n = 3 per substance and concentration