Ampicillin Effects on the Ciliary Beat Frequency of Cultured Rat Middle Ear Mucosa in vitro

Bai, Shangjie; Oh, Seung Ha

Original Article

Middle Ear Diseases

Korean Journal of Audiology 2004;8(1):66-69.

Ampicillin Effects on the Ciliary Beat Frequency of Cultured Rat Middle Ear Mucosa in vitro

¹Department of Otolaryngology, College of Medicine, Yanbian University, Yanji, China
²Department of Otolaryngology, Seoul National University College of Medicine, Seoul, Korea

백서 중이점막의 섬모진동수에 대한 Ampicillin의 효과

백상걸^¹^, 오승하^²

^¹연변대학교 의과대학 이비인후과학교실
^²서울대학교 의과대학 이비인후과학교실

Keywords: Ampicillin;Ciliary beat frequency (CBF);Middle ear.

Correspondence Author：Seung Ha Oh, 110-744 Yongon-dong, Chongno-gu, Seoul, Korea
Tel) (02) 2072-2442, Fax) (02) 745-2387, E-mail：shaoh@snu.ac.kr

Introduction

Since the middle ear mucosa is an extension and a modification of respiratory mucosa, it is conceivable that ciliary activity plays an important role in the clearance of the tympanic cavity. However, few studies on the effect of antibiotics on the ciliary activity have been published. Takeyama et al.,¹⁾ reported that erythromycin and roxithromycin increased the ciliary beat frequency (CBF) of rabbit tracheal epithelium in a dose-dependent fashion, whereas clarithromycin had no effect, and they suggested that prostaglandin and cAMP dependent regulatory pathways may be involved in this increased CBF. It was also demonstrated that ciprofloxacin also increases ciliary activity of rabbit tracheal epithelium in a concentration dependent manner.²⁾
Ampicillin has been widely used for middle ear infection. However, the direct effects of ampicillin on CBF in the middle ear epithelium have not been studied. In relation with effects of ampicillin on ciliary activity, Toremalm et al.,³⁾ reported that 161 mM ampicillin decreased CBF significantly in rabbit tracheal epithelium, and explained that the inhibitory effect of ampicillin at pH9.5 could have been a pH effect. However, in their study the concentration of ampicillin used was about 10,000 fold higher than its therapeutic concentration (6 μg/mL) and the pH effect could not be actually proved. In this study we aimed to investigate whether ampicillin with its therapeutic concentration affects ciliary beating in rat middle ear epithelium in vitro.

Materials and Methods

Materials and cell preparation

Ten 4-month-old healthy Sprague-Dawley rats were used. Their weights ranged from 220 to 235 gram. Each bulla epithelium was dissected from the underlying bone, cut into 6 small pieces, rinsed with normal saline and then incubated in DMEM-12 (Gibco BRL, Grand Island, NY) for an hour. Five different concentrations of ampicillin (Sigma, St. Louis, MO) at 0.06, 0.6, 6, 60 and 600 μg/ml were prepared in DMEM-F12. A piece of middle ear epithelium was incubated in a specially designed sterile culture dish and the temperature of which was maintained at 37℃ throughout the experiment.

Measurement of CBF

The CBF of the middle ear epithelial cells was measured using the video-computerized analysis system as described by Min YG et al..⁴⁾ Each of the 10 tissue samples was used for 1 control and 5 conditions of ampicillin treatment at 5 different concentrations. To evaluate the effect of ampicillin, CBF was measured at each concentration before and 5, 10, 20, 40, and 60 minutes after adding the ampicillin-containing medium. CBFs were measured in a selected field in each specimen serially. An average CBF value from 10 specimens was taken for each concentration studied. The mean CBF of the control and ampicillin groups were compared using the Wilcoxon rank sum test.
To ensure that ampicillin had a direct effect on CBF, ampicillin-containing medium was replaced with ampicillin-free medium after 60 mins of exposure to ampicillin at 6 or 60 μg/ml (n=2 at each concentration). We also exposed epithelia to 161 mM (approximately 60,000 μg/ml) ampicillin-contaning and pH 9.5 media for 90 minutes, to determine whether the CBF reduces significantly in rat middle ear epithelium on increasing pH as described in Toremalm's report (n=2 in each experiment).

Results

Effects of ampicillin on CBF

Exposure of ciliated rat middle ear epithelial cells to ampicillin-containing media resulted in a significant increase in CBF compare to the control media. The significant increase in CBF was detected after incubating 5 minutes in 600 μg/ml, 10 minute in 60 μg/ml, and 20 minutes in 6 μg/ml, and increase of CBF was persisted at least 60 minutes (p<0.01)(Fig. 1). Moreover, level of increase in CBF showed concentration-dependent manner over the range between 6 to 600 μg/ml (p<0.001)(Fig. 2).

Effects of ampicillin-free media on the ampicillin-induced stimulation of CBF

Replacing ampicillin-containing media with ampicillin-free medium, after cells had reached a CBF plateau at 60 minutes, caused the CBF to return to baseline levels (Fig. 3).

Effects of 60,000μg/ml ampicillin and pH 9.5 media on CBF

Though only two specimens were examined, the addition of 60,000μg/ml of ampicillin-containing medium (pH=8.3) increased CBF and this was maintained at up to 90 minutes. However, this CBF increase was much smaller than that induced by 600 μg/ml. Increasing the pH of the medium to 9.5 did not cause a significant reduction in CBF (Fig. 4).

Discussion

In this study we demonstrate that ampicillin is capable of upregulating CBF in rat middle ear epithelium. The results also suggest that this ampicillin-mediated upregulation of CBF occurs as a result of direct effect, since the replacement of an ampicillin-containing medium with an ampicillin-free medium results in a reduction of CBF to the basal level. However, further studies should be done in order to confirm whether the observed effects of ampicillin reflect an actual improvement of mucociliary clearance, which depends on the interplay between ciliary motility and the properties of mucus.
Our results differ from those of Toremalm et al.,³⁾ who reported that ampicillin has an inhibitory effect on CBF. They found that ampicillin reduces the CBF up to 50% by 55 minutes after exposure to 161 mM ampicillin solution (pH 9,5) in vitro. An ampicillin concentration of 161 mM is about 10,000-fold higher than its therapeutic concentration (6 μg/ml). In contrary, our study showed that ampicillin at 60,000 μg/ml increased CBF progressively and this was maintained for up to 90 minutes. In addition, we found that the pH of 60,000 μg/ml ampicillin-containing CMEM-F12 was pH=8.3 far less than pH 9.5. Because CBF is not influenced by pH per se between 7.0 to 9.0,⁵⁾ we can conclude that the stimulatory effect of ampicillin observed at 60,000 μg/ml may be the direct effect of ampicillin.
To investigate whether the inhibitory effect showed in the Toremalm's study attributes to pH of 9.5, we tested the effect of high pH on CBF. Although it is not statistically significant, we can observe the reduction in CBF (about 84%) by increasing the pH of medium to 9.5.
From above observation, we have reached to the conclusion that the inhibitory effect of ampicillin at 161 mM observed in Toremalm's study was not due to the high ampicillin concentration but rather to the high pH. The discrepancy between two study may reflect the different sample sites (trachea vs. middle ear), ampicillin formula, media used, and methods of CBF measurement.

We found that ampicillin at 6, 60 and 600 μg/ml increased CBF in a concentration-dependent manner, whereas ampicillin at 60,000 μg/ml had a minor stimulatory effect on CBF. This observation can suggest that the therapeutic level of ampicillin might have an additional clearing effect on the ciliary respiratory mucosa of middle ear. In the clinical setting of otitis media, the usage of ampicllin can potentially provide the dual effect on the mucosa such as an antimicrobial and an enhanced clearance.
With a new observing method of CBF, we can easily determined CBF changes in a serial way at the single cell level. However, it should be emphasized that the CBF is not necessarily representative to the actual clearance function of mucosa.

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