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Effect of Low-Intensity Focused Ultrasound on the Middle Ear in a Mouse Model of Acute Otitis Media
Ultrasound in Med. & Biol., Vol. 39, No. 3, pp. 413–423, 2013 Copyright Ó 2013 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
http://dx.doi.org/10.1016/j.ultrasmedbio.2012.10.003
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Original Contribution EFFECT OF LOW-INTENSITY FOCUSED ULTRASOUND ON THE MIDDLE EAR IN A MOUSE MODEL OF ACUTE OTITIS MEDIA KANAKO NODA, TAKASHI HIRANO, KENJI NODA, SATORU KODAMA, ISSEI ICHIMIYA, and MASASHI SUZUKI Department of Otolaryngology, Oita University Faculty of Medicine, Oita, Japan (Received 23 April 2012; revised 16 August 2012; in final form 2 October 2012)
Abstract—We hypothesized that low-intensity focused ultrasound (LIFU) increases vessel permeability and antibacterial drug activity in the mouse middle ear. We determined appropriate settings by applying LIFU to mouse ears with the external auditory canal filled with normal saline and performed histologic and immunohistologic examination. Acute otitis media was induced in mice with nontypable Haemophilus influenzae, and they were given ampicillin (50, 10, or 2 mg/kg) intraperitoneally once daily for 3 days with or without LIFU (1.0 W/cm2, 20% duty cycle, 30 s). In the LIFU(1) groups receiving the 2- and 10-mg/kg doses, viable bacteria counts, number of inflammatory cells and IL-1b and TNF-a levels in middle ear effusion were significantly lower than in the LIFU(2) groups on the same doses. Severity of AOM also tended to be reduced more in the LIFU(1) groups than in the LIFU(2) groups. LIFU application with antibiotics may be effective for middle ear infection. (E-mail: [email protected]) Ó 2013 World Federation for Ultrasound in Medicine & Biology. Key Words: Low-intensity focused ultrasound, Nontypable Haemophilus influenzae, Acute otitis media, Antibacterial drug.
been developed for cancer therapy (Shibaguchi at al. 2011). Low-frequency ultrasound applied to skin (sonophoresis) has been investigated to enhance the transdermal transport of various drugs (Maruani et al. 2010). Transcranial ultrasound pulses combined with a circulating ultrasound contrast agent has been used as a method to temporarily disrupt the blood–brain barrier in order to facilitate the targeted delivery of drugs to the central nervous system (Hynynen et al. 2005, 2006). Acute otitis media (AOM) is one of the most common infectious diseases in children. Current options for treatment depend mainly on the use of antibiotics, but AOM caused by b-lactamase–negative ampicillinresistant Haemophilus influenzae and penicillinresistant Streptococcus pneumoniae has increased among children (Brook and Gober 1998; Commisso et al. 2000). Most guidelines currently list high-dose amoxicillin (.45 mg/kg) as the first-line agent of choice for AOM caused by drug-resistant S. pneumoniae and H. influenzae (Block et al. 2007). However, the use of antibiotics disturbs the gastrointestinal flora, causing clinical symptoms such as diarrhea, which occurs in as many as 25% of patients. Symptoms range from mild and self-limiting to severe, particularly in Clostridium difficile infections (Beaugerie 2004). Manifestations of this infection can
INTRODUCTION Ultrasound increases intracellular uptake of drugs, macromolecules, DNA and fluorescent markers (Miller et al. 2002). The biologic effects of ultrasound are related to the generation of thermal energy, perturbation or sonoporation of cell membranes under the action of micro-convection or inertia cavitation and enhanced permeability of blood capillaries (Feril et al. 2004a, 2004b). Nonthermal ultrasound energy has been used recently for targeting and controlling drug release (Harrison et al. 1991; Nelson et al. 2002). Ultrasound is known to cause transient permeability in cell membranes, and in vitro electron microscopy studies have revealed that ultrasound causes the formation of small indentations or pores in cell membranes (Tachibana et al. 1999). Several types of ultrasound are used in various medical fields in combination with drug delivery systems. For example, sonodynamic therapy, which uses lowintensity ultrasound together with a sonosensitizer, has
Address correspondence to: Masashi Suzuki, MD, PhD, Department of Otolaryngology, Oita University Faculty of Medicine, 1-1 Idaigaoka, Hazama-machi, Yufu, Oita 879-5593, Japan. E-mail: [email protected] 413
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range from asymptomatic colonization of the gastrointestinal tract to life-threatening conditions such as pseudomembranous colitis (Wilcox 2003). Mitchell et al. (1996) identified toxigenic C. difficile in 13% of children at the conclusion of high-dose amoxicillin therapy for AOM, with a significantly higher frequency seen in children with diarrhea. These reports highlight the importance of exploring safer methods of antibiotic treatment that reduce the disturbance of the gastrointestinal flora. Recently, in vitro determinations of the time-course of killing have been used commonly to characterize the pharmacodynamics of the interaction between bacteria and antibacterial agents (Gloede et al. 2010) and the pharmacokinetic properties of a drug, such as its ability to reach its target, which is important for enhancing its antibacterial activity in local inflammation (Holford and Sheiner 1982). The pharmacokinetic properties and pharmacodynamics are characteristics of an antibacterial agent that should be considered in the development and prediction of the efficacy of the antibacterial therapy. We hypothesized that the use of low-intensity focused ultrasound (LIFU), which has been shown to enhance the delivery of drugs into cells, might enable adequate treatment of AOM with lower doses of antibiotics, thus alleviating some of the undesirable side effects observed at higher doses. We therefore examined whether LIFU increases vessel permeability and increases local antibacterial drug activity in the middle ears of mice with AOM induced by nontypable H. influenzae (NTHi).
MATERIALS AND METHODS The study was performed in accordance with the Law Concerning the Protection and Control of Animals (Law No. 105, October 1, 1973), Standards Relating to the Care and Custody of Laboratory Animals (Notification No. 9, March 27, 1980, Office of the Prime Minister) and Method for Sacrificing Laboratory Animals (Notification No. 40, July 4, 1995, Office of the Prime Minister). The animal-use protocol was approved by the Committee on Animal Experiments of Oita University (Approval No. F027003, January 4, 2006).
Animals Ninety-eight healthy male Balb/c mice, 5–6 weeks old and free of middle ear disease, were purchased from Seac Yoshitomi, Ltd. (Fukuoka, Japan). All mice were maintained in a pathogen-free facility until they were 5 weeks old, at which time they were used for experiments.
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Effect of low-intensity focused ultrasound on the middle ear tissue An incisional myringotomy was performed in the right ear to allow fluid into the middle ear. The middle ear and external auditory canal were then filled with normal saline solution, and LIFU was applied through the tympanic membrane (TM) with a 1-mm probe (Sonitron 2000, Rich-mar Corp., Inola, OK, USA). The ultrasound frequency, duty cycle and duration were fixed at 1 MHz, 20% and 30 s; and the intensity was varied (0.1, 0.5, 1.0 and 1.5 W/cm2). The left ear of each mouse was used as a control.
Preparation of the middle ear tissue for histologic examination The mice were killed by deep anesthesia by intraperitoneal injection of pentobarbital. The anesthetized mice were then perfused intracardially with physiologic saline containing 0.1% heparin, followed by 10% neutralbuffered formalin. The mice were killed 24 h after application of ultrasound, and the temporal bone was removed and fixed in 10% formalin neutral buffer solution. Tissues were sequentially dehydrated through a graded series of alcohols, cleared in xylene and embedded in paraffin.
Histologic evaluation of the effect of LIFU on the middle ear Sections of the temporal bone were stained with hematoxylin and eosin. Histologic changes associated with each set of LIFU parameters were observed by light microscopy. The thickness and degree of inflammatory cell infiltration in the middle ear mucosa were examined. The degree of vessel permeability around the middle ear was assessed by immunostaining for fibrinogen. Fibrinogen immunostaining was performed according to the conventional avidin-biotin complex method. Sections were deparaffinized, rinsed in phosphate-buffered saline (PBS), exposed to a 5% solution of normal rabbit serum and incubated with goat anti-fibrinogen (Nordic Immunologic Laboratories, Capistrano Beach, CA, USA). Sections were then flooded with biotin-conjugated rabbit anti-goat immunoglobulin (Ig) G (Vector Laboratories, Burlingame, CA, USA), and were incubated with avidin-biotin complex reagent (Vector Laboratories) for 1 h and developed in 0.05% 3,3’-diaminobenzidine-0.01% H2O2 substrate medium in 0.1 M phosphate buffer (DAB-H2O2). After sections were counterstained with veronal acetatebuffered 1% methyl green solution, coverslips were added and the slides were observed by light microscopy. The intensity of staining with antibody was compared between the left and right ears.
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Middle ear challenge with live nontypable NTHi and induction of experimental otitis media Strain 76 of NTHi, which was isolated from the nasopharynx of a patient with otitis media with effusion at Oita University, was used for the middle ear challenge. NTHi was grown on chocolate agar at 37 C under 5% CO2 for 16 h, and three to five clones were transferred to another plate and incubated for 4 h. The bacterial concentration was determined by optical density at a wavelength of 600 nm, and a bacterial suspension of 106 CFU/mL in PBS was prepared and stored on ice. We confirmed the bacterial concentration by counting the colonies after overnight incubation. The bacterial suspension was injected into the right tympanic cavity as follows. After an incision was made in the submandibular skin, the right inferior bulla was exposed, and two microholes were made in the bulla with a 27-gauge needle. A micropipette was inserted into one of the holes, and 10 mL (104 CFU) of live NTHi suspension was injected slowly. Eighty-eight mice were used in the experiments. The mice were monitored otomicroscopically to confirm the presence of middle ear effusion (MEE) TM changes. Pathologic course of AOM induced by NTHi We investigated the pathologic course of AOM after the injection from day 1 to day 10. On days 1, 3, 6, 7, and 10 after injection, three mice from each group were sacrificed with deep anesthesia and decapitated. Samples of MEE were obtained by myringotomy at the time of decapitation, and the middle ears were washed with 250 mL physiologic saline. MEE samples were diluted serially with PBS, and 10 mL of the diluted samples was plated on chocolate agar. Bacterial colonies were counted after overnight incubation. Inflammatory cells in the MEE samples were counted with a hemocytometer. The samples were then centrifuged and the supernatants collected and stored at 280 C for cytokine analysis by enzyme-linked immunosorbent assay (ELISA; described below). Effect of LIFU with antibiotics on AOM On day 3 after the inoculation, the 52 mice in each dosage group were given the appropriate amount of ampicillin (50, 10, or 2 mg/kg; Wako Junyaku, Osaka, Japan) intraperitoneally in 100 mL of PBS once daily for 3 d without LIFU (1.0 W/cm2, 20% duty cycle, 30 s; Fig. 1a) or with LIFU (Fig. 1b). As all mice had MEE filling the middle ear, only the external auditory canal was filled with normal saline solution, and LIFU was applied through the TM. In this study, an incisional myringotomy was not performed because of the filling of the middle ear with MEE. This schedule of antibiotic administration has been described previously (Jauris-Heipike
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Fig. 1. Scheme of LIFU study. LIFU 5 low-intensity focused ultrasound; NTHi 5 nontypable H. influenzae; PBS 5 phosphate-buffered saline.
et al. 1997). Twenty-one control mice were given 100 mL of PBS on the same schedule. On day 8 after the inoculation, the severity of AOM was graded according to a scale developed by Giebink and Wright (1983) on the basis of the color and opacity of the TM and the MEE as follows: level 0 (normal), gray and translucent TM without MEE; level 1, gray and opaque TM with serous or mucoid MEE; and level 2, yellow and opaque TM with purulent MEE. The severity score was calculated on the basis of the level (0–2) and expressed as the mean 6 SD. After monitoring the middle ear findings, all mice were sacrificed with deep anesthesia by intraperitoneal administration of pentobarbital solution and decapitated. Samples of MEE were obtained and treated as described earlier. Bacterial colonies were counted after overnight incubation. Inflammatory cells in the MEE samples were counted with a hemocytometer. The MEE samples were centrifuged, and the supernatants were collected and stored at 280 C for cytokine analysis by ELISA (described below). The precipitants were used for real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay (described below).
Detection of cytokines in MEE by ELISA Assays for tumor necrosis factor (TNF)-a and interleukin (IL)-1b were performed with a commercially available mouse ELISA kit (R&D Systems Inc., Minneapolis, MN, USA). ELISAs were performed as specified by the manufacturer. All samples were run with negative and positive controls. Standard curves were generated from known concentrations of cytokines provided by the manufacturer. The sensitivity of these assays (i.e., the lowest concentration of cytokine detectable in MEE) was less than 5.1 pg/mL for TNF-a and less than 3 pg/mL for IL-1b. The optical densities were
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read with a microtiter ELISA plate reader (Sanko Junyaku Co. Ltd., Tokyo, Japan). Real-time RT-PCR for cytokines For the detection of IL-1b and TNF-a mRNAs, total RNA was extracted from the middle ear mucosae, which were harvested under a microscope on day 10 after the inoculation. The primers and probes for the cytokines used in this study are commercially available and were obtained from Applied Biosystems (Foster City, CA, USA). Total cellular RNA extraction and the first complementary DNA (cDNA) synthesis were performed with a commercially available RNA extraction and reverse transcription kit (Qiagen, Tokyo, Japan). The total amount of RNA used for this assay in each group was adjusted to 0.25 mg. Each cDNA sample was then used as a template in a PCR amplification mixture containing forward and reverse primers for the target cytokines, forward and reverse primers for 18S ribosomal RNA (internal control), and TaqMan Universal PCR Master Mix (Applied Biosystems). PCR amplifications for the target cytokine and internal control 18S rRNA were performed in a single well of a capped 96-well optical plate. Reaction mixtures were subjected to the following amplification scheme: 1 cycle at 50 C for 2 min and 1 cycle at 95 C for 10 min, followed by 40 cycles at 95 C for 15 s (denaturation) and 60 C for 1 min (annealing and exten-
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sion). Real-time PCR data were analyzed with Sequence Detection software, version 1.6, included with the 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA). Final quantitation was derived by the comparative threshold cycle method (Livak et al. 2001) and was reported as the fold difference relative to a calibrator cDNA (untreated middle ear mucosa used as a control) prepared in parallel with the experimental cDNAs. Statistical analysis Statistical comparison between appropriate groups was performed with an unpaired two-tailed Student’s t-test; p , 0.05 was considered significant. RESULTS Histologic evaluation of LIFU We first determined the effects of LIFU by histologic analysis. In the animals exposed to no LIFU or to 0.1 and 0.5 W/cm2 with a duty cycle of 20% for 30 s, no inflammatory cells were seen in the middle ear mucosa (Fig. 2a–c). At 1.0 W/cm2 with a duty cycle of 20% for 30 s, the middle ear mucosa appeared slightly thickened with only slight infiltration by inflammatory cells (Fig. 2d). At 1.5 W/cm2 with a duty cycle of 20% for 30 s, the middle ear mucosa became thickened with extensive infiltration by inflammatory cells (Fig. 2e).
Fig. 2. The effects of LIFU by histologic analysis (original magnification 3 400). (a–c) In the animals exposed to no LIFU or to 0.1 and 0.5 W/cm2 with a duty cycle of 20% for 30 s, no inflammatory cells were seen in the middle ear mucosa. (d) At 1.0 W/cm2 with a duty cycle of 20% for 30 s, the middle ear mucosa appeared slightly thickened with only slight infiltration by inflammatory cells. (e) At 1.5 W/cm2 with a duty cycle of 20% for 30 s, the middle ear mucosa became thickened with extensive infiltration by inflammatory cells.
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Fig. 3. The effect of LIFU on vessel permeability around the middle ear (original magnification 3 400). (a) On the control side, fibrinogen staining was weak and confined to inside the vessels. (b, c) At 0.5 W/cm2 with a duty cycle of 20% for 30 s, fibrinogen staining was slightly positive around the vessels, although no vessel disruption or extravasation was evident. (d) At 1.0 W/cm2 with a duty cycle of 20% for 30 s, fibrinogen staining was mainly positive around the vessels and middle ear mucosa. (e) At 1.5 W/cm2 with a duty cycle of 20% for 30 s, fibrinogen staining strongly extended to the subepithelial mucosa as well as the vessels and mucosal surface.
Under the same LIFU conditions, fibrinogen immunostaining was performed. On the control side, fibrinogen staining was weak and confined to inside the vessels (Fig. 3a). At 0.5 W/cm2 with a duty cycle of 20% for 30 s, fibrinogen staining was slightly positive around the vessels, although no vessel disruption or extravasation was evident (Fig. 3b, c). At 1.0 W/cm2 with a duty cycle of 20% for 30 s, fibrinogen staining was positive mainly around the vessels and middle ear mucosa (Fig. 3d). At 1.5 W/cm2 with a duty cycle of 20% for 30 s, strong fibrinogen staining extended to the subepithelial mucosa as well as the vessels and mucosal surface (Fig. 3e). The presence of extravascular fibrinogen in the middle ear mucosa suggests disruption of the vessel wall caused by LIFU, although this could not be detected histologically on the sections stained with hematoxylin and eosin.
Pathologic course of AOM induced by NTHi From days 1–10 after inoculation with NTHi, all the mice had purulent MEE (level 3). Bacteria counts in the MEE samples were not different among the mice at each time point, although the number of inflammatory cells gradually increased throughout the duration of the experiments (Fig. 4a, b). The concentrations of proinflammatory cytokines IL-1b and TNF-a in the MEE
were also not different among the mice at each time point throughout the duration of the experiments (Fig. 5). Severity of AOM The antibacterial effects of ampicillin increased directly with the size of the dose. The AOM grading (0–2) was more severe in the LIFU(2) groups given 10 and 50 mg/kg ampicillin than in the corresponding LIFU(1) groups given equivalent doses (Fig. 6); however, the difference in AOM severity scores between the LIFU(2) and LIFU(1) groups did not reach statistical significance (Table 1). Effect of LIFU on bacteria counts and inflammatory cells in MEE The numbers of viable bacteria and inflammatory cells in MEE samples in all the LIFU(1) groups were lower than those in the LIFU(2) groups except for the PBS group (Fig. 7a, b). In addition, the inflammatory changes in the middle ear, such as changes in mucosal thickness and inflammatory cell infiltration, were less prominent in the LIFU group than in the control group (data not shown). The bacterial counts in the MEE samples were also significantly lower in the LIFU(1)/10 mg and 2 mg/kg ampicillin groups than in the LIFU(2) group (mean bacterial count 6 SD in
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Fig. 4. Bacteria counts and inflammatory cells in MEE. Bacteria counts in MEEs were not different among the mice each time point (a) although the number of inflammatory cells gradually increased throughout the duration of the experiments (b).
LIFU[2] vs. LIFU[1], respectively: 10 mg, 5.9 6 0.6 vs. 2.2 6 2.5, p 5 0.003; 2 mg, 6.0 6 0.8 vs. 3.7 6 2.6, p 5 0.03). The numbers of inflammatory cells in the samples were also significantly lower in the LIFU(1)/10 mg/kg ampicillin group than in the LIFU(2) group (mean severity score 6 SD in LIFU[2] vs. LIFU[1], respec-
tively: 5.9 6 0.3 vs. 5.0 6 0.6, p 5 0.003). There was no significant difference between the LIFU(1)/PBS (no ampicillin) and LIFU(2)/PBS mice in the number of inflammatory cells and viable bacteria counts. In all LIFU(1) mice, the antibacterial effects of ampicillin were dose dependent.
Fig. 5. The concentrations of IL-1b and TNF-a. The concentrations of IL-1b and TNF-a, such as proinflammatory cytokines, in MEEs were also not different among the mice at each time point throughout the duration of the experiments. IL-1b 5 interleukin 1b; TNF-a 5 tumor necrosis factor.
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Fig. 6. The effect of LIFU with antibiotics on the AOM grading. Antibacterial effects of ampicillin increased directly with the size of the dose. The AOM grading (0–2) was more severe in the LIFU(2) groups given 10 and 50 mg/kg ampicillin than in the corresponding LIFU(1) groups given equivalent doses. PBS 5 phosphate-buffered saline.
Effect of LIFU on IL-1b and TNF-a levels in MEE and IL-1b and TNF-a mRNA expression in inflammatory cells According to the ELISA results, the levels of IL-1b and TNF-a in MEE samples in all the LIFU(1) groups were lower than those in all the LIFU(2) groups. The differences in the concentration of IL-1b reached statistical significance in the groups treated with 10 mg/kg ampicillin (mean concentration of IL-1b 6 SD in LIFU[2] vs. LIFU[1], respectively: 236 6 174 vs. 42 6 70, p 5 0.003; Fig. 8a). The concentrations of TNF-a in MEE samples were also significantly lower in the LIFU(1) groups treated with 10 mg/kg and 2 mg/kg ampicillin than in the LIFU(2) groups (mean concentration of TNF-a 6 SD in LIFU[2] vs. LIFU[1], respectively: 10 mg, 68 6 54 vs. 12 6 22, p 5 0.017; 2 mg, 90 6 48 vs. 45 6 37, p 5 0.048; Fig. 8b). There was no difference in these levels between the LIFU(1)/PBS and LIFU(2)/PBS mice. According to the measurement of mRNA, IL-1b and TNF-a mRNA expression levels did not differ significantly between most LIFU(1) mice and the Table 1. The severity score of acute otitis media* Solution
LIFU(1)
LIFU(2)
PBS 2 mg of ABPC 10 mg of ABPC 50 mg of ABPC
1.9 6 0.3 1.4 6 0.5 0.8 6 0.9 0.6 6 0.6
1.7 6 0.5 1.4 6 0.5 1.1 6 0.6 1.0 6 0.8
ABPC 5 ampicillin; LIFU 5 low-intensity focused ultrasound; PBS 5 phosphate-buffered saline. * Nine mice from each group were monitored otomicroscopically at day 8 after injection with nontypeable Haemophilus influenzae. Severity score was measured according to the characteristics of middle ear effusions (MEEs) as follows: 0, no MEE; 1, serous or mucoid MEE; 2, purulent MEE. The severity score expressed as the mean 6 SD.
LIFU(2) mice that received the same ampicillin dose (Fig. 9). However, expression levels of both cytokines were slightly lower in the LIFU(1)/10 mg/kg ampicillin group than in the LIFU(2)/10 mg/kg ampicillin group. DISCUSSION Ultrasound can be classified generally as highintensity focused ultrasound (HIFU) or LIFU (Tachibana et al. 1999). Although the definitions of these categories have not been standardized, an intensity greater than 10 W/cm2 is usually designated as HIFU and that of 0.3–3.0 W/cm2 as LIFU (Shuto et al. 2006). The HIFU technique is used to treat tumors of the liver, prostate, and other sites, and this technique relies on the delivery of the energy required to raise the tissue temperature to a cytotoxic level quickly enough to prevent the tissue vasculature from having a significant effect on the extent of cell death (Uchida et al. 2011). In contrast, cell damage induced by LIFU is far less extensive than that induced by HIFU (Deng et al. 2004). Hence, LIFU produces transient permeability of cell membranes, thereby allowing foreign molecules to enter the cell. Accordingly, LIFU is commonly used to increase the effectiveness of almost all gene transfer techniques and is effective both in vitro (Ohta et al. 2003) and in vivo (Taniyama et al. 2002). Lowintensity ultrasonic therapy is becoming an important research area of ultrasonic medicine, and it has been reported that low-intensity ultrasound enhances the bactericidal action of antibiotics against bacteria in vitro and in vivo, including planktonic bacteria, bacteria in biofilms, Chlamydia organisms, and bacteria in implants (Yu et al. 2012).
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Fig. 7. The effect of LIFU on bacteria counts and inflammatory cells in MEE. The bacterial counts in MEEs were also significantly lower in the LIFU(1)/10 mg and 2 mg/kg ampicillin group than in the LIFU(2) group (mean bacterial count 6 SD in LIFU(2) vs. LIFU(1), respectively; 10 mg, 5.9 6 0.6 vs. 2.2 6 2.5, p 5 0.003; 2 mg, 6.0 6 0.8 vs. 3.7 6 2.6, p 5 0.03). The numbers of inflammatory cells in MEEs were also significantly lower in the LIFU(1)/ 10 mg/kg ampicillin group than in the LIFU(2) group (mean severity score 6 SD in LIFU[2] vs. LIFU[1], respectively; 5.9 6 0.3 vs. 5.0 6 0.6, p 5 0.003). LIFU 5 low-intensity focused ultrasound; PBS 5 phosphate-buffered saline.
The biologic effects of LIFU depend in part on the intensity and frequency used, the duration of exposure and the type of tissue through which the ultrasound wave passes or is absorbed. In short, exposure time and irradiation method are important; a longer duration of ultrasound application might be expected to increase the delivery efficiency. However, after a certain time, additional ultrasound exposure increases the likelihood of undesirable and irreversible cell damage, eventually leading to cell death (Hua et al. 2005). It is especially important to avoid unwanted effects such as mechanical and thermal damage to tissue caused by prolonged exposure (Barnett et al. 1994). This study was designed to explore the applicability of LIFU to antibiotic drug delivery in the middle ear mucosa. The ultrasound frequency, duty cycle, and duration were fixed at 1 MHz, 20%, and 30 s in accordance with the findings of previous studies (Hua et al. 2005; Inagaki et al. 2006; Shimamura et al. 2005; Shuto et al. 2006). Under this condition, we explored the optimal intensity of ultrasound by testing intensities of 0.1, 0.5, 1.0, and 1.5 W/cm2. Histologic and immunohistochemical staining showed that increasing LIFU intensities were associated with increasing amounts of mucosal thickening, inflammatory cell infiltration, and extravasation of fibrin-
ogen. Our results showed that LIFU at 1.0W/cm2 with a duty cycle of 20% for 30 s was the maximum setting that could be used without causing histologically evident damage in the mouse middle ear mucosa. In addition, fibrinogen staining was seen around the vessels and middle ear mucosa under this condition. We coupled these LIFU effects with varying doses of ampicillin, which showed a dose-dependent antibacterial effect, as expected. The number of inflammatory cells and viable bacteria, and the levels of IL-1b and TNF-a in MEE were significantly lower in the LIFU(1) group treated with 10 mg/kg ampicillin than in the LIFU(2) group treated with the same ampicillin dose. Furthermore, the number of viable bacteria and levels of TNFa in MEE were significantly lower in the LIFU(1) group treated with 2 mg/kg ampicillin than in the LIFU(2) group treated with the same ampicillin dose. However, these outcome variables did not differ significantly between LIFU(1) and LIFU(2) groups that were treated with the higher ampicillin doses (50 mg/kg) or with no ampicillin (i.e., with PBS). A likely explanation for these findings is that an ampicillin dose of 50 mg/kg is so extreme that it achieves complete or sufficient tissue delivery, regardless of other factors such as LIFU. In addition, there was a trend toward improvement in the
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Fig. 8. The effect of LIFU on IL-1b and TNF-a levels in MEEs. (a) The differences in the concentration of IL-1b reached statistical significance in the groups treated with 10 mg/kg ampicillin (mean concentration of IL-1b 6 SD in LIFU[2] vs. LIFU(1), respectively; 236 6 174 vs. 42 6 70, p 5 0.003). (b) The concentration of TNF-a in MEEs were also significantly lower in the LIFU(1)/10 mg and 2 mg/kg ampicillin group than in the LIFU(2) group (mean concentration of TNF-a 6 SD in LIFU[2] vs. LIFU(1), respectively; 10 mg, 68 6 54 vs. 12 6 22, p 5 0.017; 2 mg, 90 6 48 vs. 45 6 37, p 5 0.048). LIFU 5 low-intensity focused ultrasound; PBS 5 phosphate-buffered saline.
severity of AOM in the LIFU(1) groups compared with the LIFU(2) groups; however, the difference in AOM severity scores did not achieve statistical significance between the two groups at any dose of ampicillin. It is known that middle ear findings used to determine the severity of AOM are affected by endotoxin, which is a component of the outer membrane of all gramnegative bacteria and consists of lipo-oligosaccharide complexed with outer membrane proteins of NTHi. It has been reported that endotoxin is present in MEE of up to 80% of patients who suffer from chronic otitis media with effusion and that 67% of culture-negative MEE samples contain endotoxin (DeMaria et al. 1984). Endotoxin is also a potent inducer of inflammation and a modulator of immune responses in the middle ear (Willett et al. 1998). Moreover, administration of endotoxin alone into the middle ear induces otitis media with effusion in animals (DeMaria and Murwin 1997; Watanabe et al. 2001). These results indicate that the
severity of AOM as determined by MEE findings might be affected by not only viable bacteria but also bacterial components such as endotoxin and outer membrane proteins. As these results, the difference between the two groups in the AOM severity score was not statistically significant in this study. In previous studies, extravasation of fibrinogen was used as a sensitive marker to show vessel disruption in the cochlea and submandibular gland (Ichimiya et al. 1999; Shuto et al. 2006). Shuto et al. (2006) reported that immunostaining after LIFU application showed the presence of extravascular fibrinogen in the submandibular gland, and that disruption of the vessel wall continued for at least 5 d after LIFU application. Our findings indicated that the application of LIFU to exposed mouse middle ear mucosa produced transient disruption of the vessels, allowing fibrinogen to diffuse from the vessels for several days. The molecular weight of murine fibrinogen (soluble dimer) is 340 kDa. It may be that drugs of
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Fig. 9. The effect of LIFU on IL-1b and TNF-a mRNA expression in inflammatory cells. The IL-1b mRNA (a) and TNFa mRNA (b) expression levels did not differ significantly between most LIFU(1) mice and the LIFU(2) mice that received the same ampicillin dose. However, both cytokine expression levels were slightly lower in the LIFU(1)/ 10 mg/kg ampicillin group than in the LIFU(2)/10 mg/kg ampicillin group. LIFU 5 low-intensity focused ultrasound; PBS 5 phosphate-buffered saline.
similar molecular weight can pass through the vessels to enter target organs; this has considerable clinical implications and can be particularly applicable to various antibiotic agents. The results from Yu et al. (2012) also support ours, and they reported that low-intensity ultrasound alone is not effective in killing bacteria, whereas the combination of low intensity ultrasound and antibiotics is promising. He et al. (2011) also reported the effectiveness of LIFU with microbubbles on biofilms of Streptococcus epidermidis. Biofilm densities and the viable counts of bacteria recovered from the biofilm were significantly decreased in biofilms treated with vancomycin and microbubbles combined with 1.0 W/ cm2 LIFU. Ours is the first report in which LIFU with antibiotic treatment is shown to be effective in enhancing bacterial clearance and reducing inflammatory responses in the middle ear with AOM. LIFU application along with antibiotics may be an effective strategy for the treatment of AOM, although further study is
needed to estimate the amount of acoustic energy being delivered to the middle ear to confirm the effect of LIFU and also to protect the inner ear against damage to move closer to establishing LIFU clinical trials in human patients with AOM. CONCLUSION Our results show that in vivo LIFU can disrupt vessels and increase vessel permeability in the mouse middle ear mucosa without causing tissue damage. The first part of this study showed the maximum LIFU parameters that would not cause histologic damage in the murine middle ear mucosa: 1.0 W/cm2 with a duty cycle of 20% for 30 s. When ampicillin was given for 3 d at intraperitoneal doses of 2 and 10 mg/kg, thenumber of inflammatory cells, viable bacteria counts, and levels of IL-1b and TNF-a in MEEs were significantly lower when LIFU was administered to the
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ear than when it was not. LIFU application along with antibiotics may be an effective strategy for treatment of AOM. Acknowledgments—This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan (22591885). We thank A. Iwamoto in our department for assistance.
REFERENCES Barnett SB, Ter Haar GR, Ziskin MC, Nyborg WL, Maeda K, Bang J. Current status of research on biophysical effects of ultrasound. Ultrasound Med Biol 1994;20:205–218. Beaugerie L. Antibiotic-associated diarrhoea. Best Pract Res Clin Gastroenterol 2004;18:337–352. Brook I, Gober AE. Microbiologic characteristics of persistent otitis media. Arch Otolaryngol Head Neck Surg 1998;124: 1350–1352. Commisso R, Romero-Orellano F, Montanaro PB, Romero-Moroni F, Romero-Diaz R. Acute otitis media: bacteriology and bacterial resistance in 205 pediatric patients. Int J Pediatr Otorhinolaryngol 2000; 56:23–31. DeMaria TF, Murwin DM. Tumor necrosis factor during experimental lipopolysaccharide-induced otitis media. Laryngoscope 1997;107: 369–372. DeMaria TF, Prior RB, Briggs BR, Lim DJ, Birck HG. Endotoxin in middle-ear effusions from patients with chronic otitis media with effusion. J Clin Microbiol 1984;20:15–17. Deng CX, Sieling F, Pan H, Cui J. Ultrasound-induced cell membrane porosity. Ultrasound Med Biol 2004;30:519–526. Feril LB Jr, Kondo T. Biological effects of low intensity ultrasound: the mechanism involved, and its implications on therapy and on biosafety of ultrasound. J Radiat Res 2004a;45:479–489. Feril LB Jr, Kondo T, Takaya K, Riesz P. Enhanced ultrasound induced apoptosis and cell lysis by a hypotonic medium. Int J Radiat Biol 2004b;80:165–175. Giebink GS, Wright PF. Different virulence of Influenza A virus strains and susceptibility to pneumococcal otitis media in chinchillas. Infect Immun 1983;41:913–920. Gloede J, Scheerans C, Derendorf H, Kloft C. In vitro pharmacodynamic models to determine the effect of antibacterial drugs. J Antimicrob Chemother 2010;65:186–201. Harrison GH, Balcer-Kubiczek EK, Eddy HA. Potentiation of chemotherapy by low-level ultrasound. Int J Radiat Biol 1991;59: 1453–1466. He N, Hu J, Liu H, Zhu T, Huang B, Wang X, Wu Y, Wang W, Qu D. Enhancement of vancomycin activity against biofilms by using ultrasound-targeted microbubble destruction. Antimicrob Agents Chemother 2011;55:5331–5337. Holford NH, Sheiner LB. Kinetics of pharmacologic response. Pharmacol Ther 1982;16:143–166. Hua P, Yun Z, Olivier I, Jianmin C, Cheri XD. Study of sonoporation dynamics affected by ultrasound duty cycle. Ultrasound Med Biol 2005;31:849–856. Hynynen K, McDannold N, Vykhodtseva N, Raymond S, Weissleder R, Jolesz FA, Sheikov N. Focal disruption of the blood–brain barrier due to 260-kHz ultrasound bursts: a method for molecular imaging and targeted drug delivery. J Neurosurgery 2006;105:445–454. Hynynen K, McDannold N, Sheikov NA, Jolesz FA, Vykhodtseva N. Local and reversible blood-brain barrier disruption by noninvasive
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focused ultrasound at frequencies suitable for trans-skull sonications. Neuroimage 2005;24:12–20. Ichimiya I, Suzuki M, Hirano T, Mogi G. The influence of pneumococcal otitis media on the cochlear lateral wall. Hear Res 1999; 131:128–134. Inagaki H, Suzuki J, Ogawa M, Taniyama Y, Morishita R, Isobe M. Ultrasound-microbubble mediated NF-kb decoy transfection attenuates neo-intimal formation after arterial injury to mice. J Vasc Res 2006;43:12–18. Jauris-Heipike S, Leake ER, Billy JM, DeMaria TF. The effect of antibiotic treatment on the release of endotoxin during nontypeable Haemophilus influenza-induced otitis media in the chinchilla. Acta Otolaryngol 1997;117:109–112. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 22DDCT method. Method 2001;25:402–408. Maruani A, Vierron E, Machet L, Giraudeau B, Boucaud A. Efficiency of low-frequency ultrasound sonophoresis in skin penetration of histamine: a randomized study in humans. Int J Pharm 2010;385: 37–41. Miller DL, Pislaru SV, Greenleaf JE. Sonoporation: mechanical DNA delivery by ultrasonic cavitation. Somat Cell Mol Genet 2002;27: 115–134. Mitchell DK, Van R, Masson EH, Norris DM, Pickering LK. Prospective study of toxigenic Clostridium difficle in children given amoxicillin/ clavulanate for otitis media. Pediatric Infect Dis J 1996;15:514–519. Nelson JL, Roeder BL, Roloff F, Pitt WG. Ultrasonically activated chemotherapeutic drug delivery in a rat model. Cancer Res 2002; 62:7280–7283. Ohta S, Suzuki K, Tachibana K, Yamada G. Microbubble-enhanced sonoporation-efficient gene transduction technique for chick embryos. Genesis 2003;37:91–101. Shibaguchi H, Tsuru H, Kuroki M, Kuroki M. Sonodynamic cancer therapy: a non-invasive and repeatable approach using lowintensity ultrasound with a sonosensitizer. Anticancer Res 2011; 31:2425–2429. Shimamura M, Sato N, Taniyama Y, Kurinami H, Tanaka H, Takami T, Ogihara T, Tohyama M, Kaneda Y, Morishita R. Gene transfer into adult rat spinal chord using naked plasmid DNA and ultrasound microbubble. J Gene Med 2005;7:1468–1474. Shuto J, Ichimiya I, Suzuki M. Effects of low-intensity focused ultrasound on the mouse submandibular gland. Ultrasound Med Biol 2006;32:587–594. Tachibana K, Uchida T, Ogawa K, Yamashita N, Tamura K. Induction of cell-membrane porosity by ultrasound. Lancet 1999;353:1409. Taniyama Y, Tachibana K, Hiraoka K, Namba T, Yamasaki K, Hashiya N, Aoki M, Ogihara T, Yasufumi K, Morishita R. Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation 2002;105:1233–1239. Uchida T, Nakano M, Hongo S, Shoji S, Nagata Y, Satoh T, Baba S, Usui Y, Terachi T. High-intensity focused ultrasound therapy for prostate cancer. Int J Urol 2012;19:187–201. Watanabe T, Hirano T, Suzuki M, Kurono Y, Goro M. Role of interleukin-1b in a murine model of otitis media with effusion. Ann Otol Rhinol Laryngol 2001;110:574–580. Wilcox MH. Clostridium difficle infection and pseudomembranous colitis. Best Pract Res Clin Gastroenterol 2003;17:475–493. Willett DN, Rezaee RP, Billy JM, Tighe MB, DeMaria TF. Relationship of endotoxin to tumor necrosis factor-a and interleukin-1b in children with otitis media with effusion. Ann Otol Rhinol Laryngol 1998;107:28–33. Yu H, Chen S, Cao P. Synergistic bactericidal effects and mechanisms of low intensity ultrasound and antibiotics against bacteria: a review. Ultrason Sonochem 2012;19:377–382.
http://dx.doi.org/10.1016/j.ultrasmedbio.2012.10.003
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Original Contribution EFFECT OF LOW-INTENSITY FOCUSED ULTRASOUND ON THE MIDDLE EAR IN A MOUSE MODEL OF ACUTE OTITIS MEDIA KANAKO NODA, TAKASHI HIRANO, KENJI NODA, SATORU KODAMA, ISSEI ICHIMIYA, and MASASHI SUZUKI Department of Otolaryngology, Oita University Faculty of Medicine, Oita, Japan (Received 23 April 2012; revised 16 August 2012; in final form 2 October 2012)
Abstract—We hypothesized that low-intensity focused ultrasound (LIFU) increases vessel permeability and antibacterial drug activity in the mouse middle ear. We determined appropriate settings by applying LIFU to mouse ears with the external auditory canal filled with normal saline and performed histologic and immunohistologic examination. Acute otitis media was induced in mice with nontypable Haemophilus influenzae, and they were given ampicillin (50, 10, or 2 mg/kg) intraperitoneally once daily for 3 days with or without LIFU (1.0 W/cm2, 20% duty cycle, 30 s). In the LIFU(1) groups receiving the 2- and 10-mg/kg doses, viable bacteria counts, number of inflammatory cells and IL-1b and TNF-a levels in middle ear effusion were significantly lower than in the LIFU(2) groups on the same doses. Severity of AOM also tended to be reduced more in the LIFU(1) groups than in the LIFU(2) groups. LIFU application with antibiotics may be effective for middle ear infection. (E-mail: [email protected]) Ó 2013 World Federation for Ultrasound in Medicine & Biology. Key Words: Low-intensity focused ultrasound, Nontypable Haemophilus influenzae, Acute otitis media, Antibacterial drug.
been developed for cancer therapy (Shibaguchi at al. 2011). Low-frequency ultrasound applied to skin (sonophoresis) has been investigated to enhance the transdermal transport of various drugs (Maruani et al. 2010). Transcranial ultrasound pulses combined with a circulating ultrasound contrast agent has been used as a method to temporarily disrupt the blood–brain barrier in order to facilitate the targeted delivery of drugs to the central nervous system (Hynynen et al. 2005, 2006). Acute otitis media (AOM) is one of the most common infectious diseases in children. Current options for treatment depend mainly on the use of antibiotics, but AOM caused by b-lactamase–negative ampicillinresistant Haemophilus influenzae and penicillinresistant Streptococcus pneumoniae has increased among children (Brook and Gober 1998; Commisso et al. 2000). Most guidelines currently list high-dose amoxicillin (.45 mg/kg) as the first-line agent of choice for AOM caused by drug-resistant S. pneumoniae and H. influenzae (Block et al. 2007). However, the use of antibiotics disturbs the gastrointestinal flora, causing clinical symptoms such as diarrhea, which occurs in as many as 25% of patients. Symptoms range from mild and self-limiting to severe, particularly in Clostridium difficile infections (Beaugerie 2004). Manifestations of this infection can
INTRODUCTION Ultrasound increases intracellular uptake of drugs, macromolecules, DNA and fluorescent markers (Miller et al. 2002). The biologic effects of ultrasound are related to the generation of thermal energy, perturbation or sonoporation of cell membranes under the action of micro-convection or inertia cavitation and enhanced permeability of blood capillaries (Feril et al. 2004a, 2004b). Nonthermal ultrasound energy has been used recently for targeting and controlling drug release (Harrison et al. 1991; Nelson et al. 2002). Ultrasound is known to cause transient permeability in cell membranes, and in vitro electron microscopy studies have revealed that ultrasound causes the formation of small indentations or pores in cell membranes (Tachibana et al. 1999). Several types of ultrasound are used in various medical fields in combination with drug delivery systems. For example, sonodynamic therapy, which uses lowintensity ultrasound together with a sonosensitizer, has
Address correspondence to: Masashi Suzuki, MD, PhD, Department of Otolaryngology, Oita University Faculty of Medicine, 1-1 Idaigaoka, Hazama-machi, Yufu, Oita 879-5593, Japan. E-mail: [email protected] 413
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range from asymptomatic colonization of the gastrointestinal tract to life-threatening conditions such as pseudomembranous colitis (Wilcox 2003). Mitchell et al. (1996) identified toxigenic C. difficile in 13% of children at the conclusion of high-dose amoxicillin therapy for AOM, with a significantly higher frequency seen in children with diarrhea. These reports highlight the importance of exploring safer methods of antibiotic treatment that reduce the disturbance of the gastrointestinal flora. Recently, in vitro determinations of the time-course of killing have been used commonly to characterize the pharmacodynamics of the interaction between bacteria and antibacterial agents (Gloede et al. 2010) and the pharmacokinetic properties of a drug, such as its ability to reach its target, which is important for enhancing its antibacterial activity in local inflammation (Holford and Sheiner 1982). The pharmacokinetic properties and pharmacodynamics are characteristics of an antibacterial agent that should be considered in the development and prediction of the efficacy of the antibacterial therapy. We hypothesized that the use of low-intensity focused ultrasound (LIFU), which has been shown to enhance the delivery of drugs into cells, might enable adequate treatment of AOM with lower doses of antibiotics, thus alleviating some of the undesirable side effects observed at higher doses. We therefore examined whether LIFU increases vessel permeability and increases local antibacterial drug activity in the middle ears of mice with AOM induced by nontypable H. influenzae (NTHi).
MATERIALS AND METHODS The study was performed in accordance with the Law Concerning the Protection and Control of Animals (Law No. 105, October 1, 1973), Standards Relating to the Care and Custody of Laboratory Animals (Notification No. 9, March 27, 1980, Office of the Prime Minister) and Method for Sacrificing Laboratory Animals (Notification No. 40, July 4, 1995, Office of the Prime Minister). The animal-use protocol was approved by the Committee on Animal Experiments of Oita University (Approval No. F027003, January 4, 2006).
Animals Ninety-eight healthy male Balb/c mice, 5–6 weeks old and free of middle ear disease, were purchased from Seac Yoshitomi, Ltd. (Fukuoka, Japan). All mice were maintained in a pathogen-free facility until they were 5 weeks old, at which time they were used for experiments.
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Effect of low-intensity focused ultrasound on the middle ear tissue An incisional myringotomy was performed in the right ear to allow fluid into the middle ear. The middle ear and external auditory canal were then filled with normal saline solution, and LIFU was applied through the tympanic membrane (TM) with a 1-mm probe (Sonitron 2000, Rich-mar Corp., Inola, OK, USA). The ultrasound frequency, duty cycle and duration were fixed at 1 MHz, 20% and 30 s; and the intensity was varied (0.1, 0.5, 1.0 and 1.5 W/cm2). The left ear of each mouse was used as a control.
Preparation of the middle ear tissue for histologic examination The mice were killed by deep anesthesia by intraperitoneal injection of pentobarbital. The anesthetized mice were then perfused intracardially with physiologic saline containing 0.1% heparin, followed by 10% neutralbuffered formalin. The mice were killed 24 h after application of ultrasound, and the temporal bone was removed and fixed in 10% formalin neutral buffer solution. Tissues were sequentially dehydrated through a graded series of alcohols, cleared in xylene and embedded in paraffin.
Histologic evaluation of the effect of LIFU on the middle ear Sections of the temporal bone were stained with hematoxylin and eosin. Histologic changes associated with each set of LIFU parameters were observed by light microscopy. The thickness and degree of inflammatory cell infiltration in the middle ear mucosa were examined. The degree of vessel permeability around the middle ear was assessed by immunostaining for fibrinogen. Fibrinogen immunostaining was performed according to the conventional avidin-biotin complex method. Sections were deparaffinized, rinsed in phosphate-buffered saline (PBS), exposed to a 5% solution of normal rabbit serum and incubated with goat anti-fibrinogen (Nordic Immunologic Laboratories, Capistrano Beach, CA, USA). Sections were then flooded with biotin-conjugated rabbit anti-goat immunoglobulin (Ig) G (Vector Laboratories, Burlingame, CA, USA), and were incubated with avidin-biotin complex reagent (Vector Laboratories) for 1 h and developed in 0.05% 3,3’-diaminobenzidine-0.01% H2O2 substrate medium in 0.1 M phosphate buffer (DAB-H2O2). After sections were counterstained with veronal acetatebuffered 1% methyl green solution, coverslips were added and the slides were observed by light microscopy. The intensity of staining with antibody was compared between the left and right ears.
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Middle ear challenge with live nontypable NTHi and induction of experimental otitis media Strain 76 of NTHi, which was isolated from the nasopharynx of a patient with otitis media with effusion at Oita University, was used for the middle ear challenge. NTHi was grown on chocolate agar at 37 C under 5% CO2 for 16 h, and three to five clones were transferred to another plate and incubated for 4 h. The bacterial concentration was determined by optical density at a wavelength of 600 nm, and a bacterial suspension of 106 CFU/mL in PBS was prepared and stored on ice. We confirmed the bacterial concentration by counting the colonies after overnight incubation. The bacterial suspension was injected into the right tympanic cavity as follows. After an incision was made in the submandibular skin, the right inferior bulla was exposed, and two microholes were made in the bulla with a 27-gauge needle. A micropipette was inserted into one of the holes, and 10 mL (104 CFU) of live NTHi suspension was injected slowly. Eighty-eight mice were used in the experiments. The mice were monitored otomicroscopically to confirm the presence of middle ear effusion (MEE) TM changes. Pathologic course of AOM induced by NTHi We investigated the pathologic course of AOM after the injection from day 1 to day 10. On days 1, 3, 6, 7, and 10 after injection, three mice from each group were sacrificed with deep anesthesia and decapitated. Samples of MEE were obtained by myringotomy at the time of decapitation, and the middle ears were washed with 250 mL physiologic saline. MEE samples were diluted serially with PBS, and 10 mL of the diluted samples was plated on chocolate agar. Bacterial colonies were counted after overnight incubation. Inflammatory cells in the MEE samples were counted with a hemocytometer. The samples were then centrifuged and the supernatants collected and stored at 280 C for cytokine analysis by enzyme-linked immunosorbent assay (ELISA; described below). Effect of LIFU with antibiotics on AOM On day 3 after the inoculation, the 52 mice in each dosage group were given the appropriate amount of ampicillin (50, 10, or 2 mg/kg; Wako Junyaku, Osaka, Japan) intraperitoneally in 100 mL of PBS once daily for 3 d without LIFU (1.0 W/cm2, 20% duty cycle, 30 s; Fig. 1a) or with LIFU (Fig. 1b). As all mice had MEE filling the middle ear, only the external auditory canal was filled with normal saline solution, and LIFU was applied through the TM. In this study, an incisional myringotomy was not performed because of the filling of the middle ear with MEE. This schedule of antibiotic administration has been described previously (Jauris-Heipike
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Fig. 1. Scheme of LIFU study. LIFU 5 low-intensity focused ultrasound; NTHi 5 nontypable H. influenzae; PBS 5 phosphate-buffered saline.
et al. 1997). Twenty-one control mice were given 100 mL of PBS on the same schedule. On day 8 after the inoculation, the severity of AOM was graded according to a scale developed by Giebink and Wright (1983) on the basis of the color and opacity of the TM and the MEE as follows: level 0 (normal), gray and translucent TM without MEE; level 1, gray and opaque TM with serous or mucoid MEE; and level 2, yellow and opaque TM with purulent MEE. The severity score was calculated on the basis of the level (0–2) and expressed as the mean 6 SD. After monitoring the middle ear findings, all mice were sacrificed with deep anesthesia by intraperitoneal administration of pentobarbital solution and decapitated. Samples of MEE were obtained and treated as described earlier. Bacterial colonies were counted after overnight incubation. Inflammatory cells in the MEE samples were counted with a hemocytometer. The MEE samples were centrifuged, and the supernatants were collected and stored at 280 C for cytokine analysis by ELISA (described below). The precipitants were used for real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay (described below).
Detection of cytokines in MEE by ELISA Assays for tumor necrosis factor (TNF)-a and interleukin (IL)-1b were performed with a commercially available mouse ELISA kit (R&D Systems Inc., Minneapolis, MN, USA). ELISAs were performed as specified by the manufacturer. All samples were run with negative and positive controls. Standard curves were generated from known concentrations of cytokines provided by the manufacturer. The sensitivity of these assays (i.e., the lowest concentration of cytokine detectable in MEE) was less than 5.1 pg/mL for TNF-a and less than 3 pg/mL for IL-1b. The optical densities were
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read with a microtiter ELISA plate reader (Sanko Junyaku Co. Ltd., Tokyo, Japan). Real-time RT-PCR for cytokines For the detection of IL-1b and TNF-a mRNAs, total RNA was extracted from the middle ear mucosae, which were harvested under a microscope on day 10 after the inoculation. The primers and probes for the cytokines used in this study are commercially available and were obtained from Applied Biosystems (Foster City, CA, USA). Total cellular RNA extraction and the first complementary DNA (cDNA) synthesis were performed with a commercially available RNA extraction and reverse transcription kit (Qiagen, Tokyo, Japan). The total amount of RNA used for this assay in each group was adjusted to 0.25 mg. Each cDNA sample was then used as a template in a PCR amplification mixture containing forward and reverse primers for the target cytokines, forward and reverse primers for 18S ribosomal RNA (internal control), and TaqMan Universal PCR Master Mix (Applied Biosystems). PCR amplifications for the target cytokine and internal control 18S rRNA were performed in a single well of a capped 96-well optical plate. Reaction mixtures were subjected to the following amplification scheme: 1 cycle at 50 C for 2 min and 1 cycle at 95 C for 10 min, followed by 40 cycles at 95 C for 15 s (denaturation) and 60 C for 1 min (annealing and exten-
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sion). Real-time PCR data were analyzed with Sequence Detection software, version 1.6, included with the 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA). Final quantitation was derived by the comparative threshold cycle method (Livak et al. 2001) and was reported as the fold difference relative to a calibrator cDNA (untreated middle ear mucosa used as a control) prepared in parallel with the experimental cDNAs. Statistical analysis Statistical comparison between appropriate groups was performed with an unpaired two-tailed Student’s t-test; p , 0.05 was considered significant. RESULTS Histologic evaluation of LIFU We first determined the effects of LIFU by histologic analysis. In the animals exposed to no LIFU or to 0.1 and 0.5 W/cm2 with a duty cycle of 20% for 30 s, no inflammatory cells were seen in the middle ear mucosa (Fig. 2a–c). At 1.0 W/cm2 with a duty cycle of 20% for 30 s, the middle ear mucosa appeared slightly thickened with only slight infiltration by inflammatory cells (Fig. 2d). At 1.5 W/cm2 with a duty cycle of 20% for 30 s, the middle ear mucosa became thickened with extensive infiltration by inflammatory cells (Fig. 2e).
Fig. 2. The effects of LIFU by histologic analysis (original magnification 3 400). (a–c) In the animals exposed to no LIFU or to 0.1 and 0.5 W/cm2 with a duty cycle of 20% for 30 s, no inflammatory cells were seen in the middle ear mucosa. (d) At 1.0 W/cm2 with a duty cycle of 20% for 30 s, the middle ear mucosa appeared slightly thickened with only slight infiltration by inflammatory cells. (e) At 1.5 W/cm2 with a duty cycle of 20% for 30 s, the middle ear mucosa became thickened with extensive infiltration by inflammatory cells.
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Fig. 3. The effect of LIFU on vessel permeability around the middle ear (original magnification 3 400). (a) On the control side, fibrinogen staining was weak and confined to inside the vessels. (b, c) At 0.5 W/cm2 with a duty cycle of 20% for 30 s, fibrinogen staining was slightly positive around the vessels, although no vessel disruption or extravasation was evident. (d) At 1.0 W/cm2 with a duty cycle of 20% for 30 s, fibrinogen staining was mainly positive around the vessels and middle ear mucosa. (e) At 1.5 W/cm2 with a duty cycle of 20% for 30 s, fibrinogen staining strongly extended to the subepithelial mucosa as well as the vessels and mucosal surface.
Under the same LIFU conditions, fibrinogen immunostaining was performed. On the control side, fibrinogen staining was weak and confined to inside the vessels (Fig. 3a). At 0.5 W/cm2 with a duty cycle of 20% for 30 s, fibrinogen staining was slightly positive around the vessels, although no vessel disruption or extravasation was evident (Fig. 3b, c). At 1.0 W/cm2 with a duty cycle of 20% for 30 s, fibrinogen staining was positive mainly around the vessels and middle ear mucosa (Fig. 3d). At 1.5 W/cm2 with a duty cycle of 20% for 30 s, strong fibrinogen staining extended to the subepithelial mucosa as well as the vessels and mucosal surface (Fig. 3e). The presence of extravascular fibrinogen in the middle ear mucosa suggests disruption of the vessel wall caused by LIFU, although this could not be detected histologically on the sections stained with hematoxylin and eosin.
Pathologic course of AOM induced by NTHi From days 1–10 after inoculation with NTHi, all the mice had purulent MEE (level 3). Bacteria counts in the MEE samples were not different among the mice at each time point, although the number of inflammatory cells gradually increased throughout the duration of the experiments (Fig. 4a, b). The concentrations of proinflammatory cytokines IL-1b and TNF-a in the MEE
were also not different among the mice at each time point throughout the duration of the experiments (Fig. 5). Severity of AOM The antibacterial effects of ampicillin increased directly with the size of the dose. The AOM grading (0–2) was more severe in the LIFU(2) groups given 10 and 50 mg/kg ampicillin than in the corresponding LIFU(1) groups given equivalent doses (Fig. 6); however, the difference in AOM severity scores between the LIFU(2) and LIFU(1) groups did not reach statistical significance (Table 1). Effect of LIFU on bacteria counts and inflammatory cells in MEE The numbers of viable bacteria and inflammatory cells in MEE samples in all the LIFU(1) groups were lower than those in the LIFU(2) groups except for the PBS group (Fig. 7a, b). In addition, the inflammatory changes in the middle ear, such as changes in mucosal thickness and inflammatory cell infiltration, were less prominent in the LIFU group than in the control group (data not shown). The bacterial counts in the MEE samples were also significantly lower in the LIFU(1)/10 mg and 2 mg/kg ampicillin groups than in the LIFU(2) group (mean bacterial count 6 SD in
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Fig. 4. Bacteria counts and inflammatory cells in MEE. Bacteria counts in MEEs were not different among the mice each time point (a) although the number of inflammatory cells gradually increased throughout the duration of the experiments (b).
LIFU[2] vs. LIFU[1], respectively: 10 mg, 5.9 6 0.6 vs. 2.2 6 2.5, p 5 0.003; 2 mg, 6.0 6 0.8 vs. 3.7 6 2.6, p 5 0.03). The numbers of inflammatory cells in the samples were also significantly lower in the LIFU(1)/10 mg/kg ampicillin group than in the LIFU(2) group (mean severity score 6 SD in LIFU[2] vs. LIFU[1], respec-
tively: 5.9 6 0.3 vs. 5.0 6 0.6, p 5 0.003). There was no significant difference between the LIFU(1)/PBS (no ampicillin) and LIFU(2)/PBS mice in the number of inflammatory cells and viable bacteria counts. In all LIFU(1) mice, the antibacterial effects of ampicillin were dose dependent.
Fig. 5. The concentrations of IL-1b and TNF-a. The concentrations of IL-1b and TNF-a, such as proinflammatory cytokines, in MEEs were also not different among the mice at each time point throughout the duration of the experiments. IL-1b 5 interleukin 1b; TNF-a 5 tumor necrosis factor.
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Fig. 6. The effect of LIFU with antibiotics on the AOM grading. Antibacterial effects of ampicillin increased directly with the size of the dose. The AOM grading (0–2) was more severe in the LIFU(2) groups given 10 and 50 mg/kg ampicillin than in the corresponding LIFU(1) groups given equivalent doses. PBS 5 phosphate-buffered saline.
Effect of LIFU on IL-1b and TNF-a levels in MEE and IL-1b and TNF-a mRNA expression in inflammatory cells According to the ELISA results, the levels of IL-1b and TNF-a in MEE samples in all the LIFU(1) groups were lower than those in all the LIFU(2) groups. The differences in the concentration of IL-1b reached statistical significance in the groups treated with 10 mg/kg ampicillin (mean concentration of IL-1b 6 SD in LIFU[2] vs. LIFU[1], respectively: 236 6 174 vs. 42 6 70, p 5 0.003; Fig. 8a). The concentrations of TNF-a in MEE samples were also significantly lower in the LIFU(1) groups treated with 10 mg/kg and 2 mg/kg ampicillin than in the LIFU(2) groups (mean concentration of TNF-a 6 SD in LIFU[2] vs. LIFU[1], respectively: 10 mg, 68 6 54 vs. 12 6 22, p 5 0.017; 2 mg, 90 6 48 vs. 45 6 37, p 5 0.048; Fig. 8b). There was no difference in these levels between the LIFU(1)/PBS and LIFU(2)/PBS mice. According to the measurement of mRNA, IL-1b and TNF-a mRNA expression levels did not differ significantly between most LIFU(1) mice and the Table 1. The severity score of acute otitis media* Solution
LIFU(1)
LIFU(2)
PBS 2 mg of ABPC 10 mg of ABPC 50 mg of ABPC
1.9 6 0.3 1.4 6 0.5 0.8 6 0.9 0.6 6 0.6
1.7 6 0.5 1.4 6 0.5 1.1 6 0.6 1.0 6 0.8
ABPC 5 ampicillin; LIFU 5 low-intensity focused ultrasound; PBS 5 phosphate-buffered saline. * Nine mice from each group were monitored otomicroscopically at day 8 after injection with nontypeable Haemophilus influenzae. Severity score was measured according to the characteristics of middle ear effusions (MEEs) as follows: 0, no MEE; 1, serous or mucoid MEE; 2, purulent MEE. The severity score expressed as the mean 6 SD.
LIFU(2) mice that received the same ampicillin dose (Fig. 9). However, expression levels of both cytokines were slightly lower in the LIFU(1)/10 mg/kg ampicillin group than in the LIFU(2)/10 mg/kg ampicillin group. DISCUSSION Ultrasound can be classified generally as highintensity focused ultrasound (HIFU) or LIFU (Tachibana et al. 1999). Although the definitions of these categories have not been standardized, an intensity greater than 10 W/cm2 is usually designated as HIFU and that of 0.3–3.0 W/cm2 as LIFU (Shuto et al. 2006). The HIFU technique is used to treat tumors of the liver, prostate, and other sites, and this technique relies on the delivery of the energy required to raise the tissue temperature to a cytotoxic level quickly enough to prevent the tissue vasculature from having a significant effect on the extent of cell death (Uchida et al. 2011). In contrast, cell damage induced by LIFU is far less extensive than that induced by HIFU (Deng et al. 2004). Hence, LIFU produces transient permeability of cell membranes, thereby allowing foreign molecules to enter the cell. Accordingly, LIFU is commonly used to increase the effectiveness of almost all gene transfer techniques and is effective both in vitro (Ohta et al. 2003) and in vivo (Taniyama et al. 2002). Lowintensity ultrasonic therapy is becoming an important research area of ultrasonic medicine, and it has been reported that low-intensity ultrasound enhances the bactericidal action of antibiotics against bacteria in vitro and in vivo, including planktonic bacteria, bacteria in biofilms, Chlamydia organisms, and bacteria in implants (Yu et al. 2012).
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Fig. 7. The effect of LIFU on bacteria counts and inflammatory cells in MEE. The bacterial counts in MEEs were also significantly lower in the LIFU(1)/10 mg and 2 mg/kg ampicillin group than in the LIFU(2) group (mean bacterial count 6 SD in LIFU(2) vs. LIFU(1), respectively; 10 mg, 5.9 6 0.6 vs. 2.2 6 2.5, p 5 0.003; 2 mg, 6.0 6 0.8 vs. 3.7 6 2.6, p 5 0.03). The numbers of inflammatory cells in MEEs were also significantly lower in the LIFU(1)/ 10 mg/kg ampicillin group than in the LIFU(2) group (mean severity score 6 SD in LIFU[2] vs. LIFU[1], respectively; 5.9 6 0.3 vs. 5.0 6 0.6, p 5 0.003). LIFU 5 low-intensity focused ultrasound; PBS 5 phosphate-buffered saline.
The biologic effects of LIFU depend in part on the intensity and frequency used, the duration of exposure and the type of tissue through which the ultrasound wave passes or is absorbed. In short, exposure time and irradiation method are important; a longer duration of ultrasound application might be expected to increase the delivery efficiency. However, after a certain time, additional ultrasound exposure increases the likelihood of undesirable and irreversible cell damage, eventually leading to cell death (Hua et al. 2005). It is especially important to avoid unwanted effects such as mechanical and thermal damage to tissue caused by prolonged exposure (Barnett et al. 1994). This study was designed to explore the applicability of LIFU to antibiotic drug delivery in the middle ear mucosa. The ultrasound frequency, duty cycle, and duration were fixed at 1 MHz, 20%, and 30 s in accordance with the findings of previous studies (Hua et al. 2005; Inagaki et al. 2006; Shimamura et al. 2005; Shuto et al. 2006). Under this condition, we explored the optimal intensity of ultrasound by testing intensities of 0.1, 0.5, 1.0, and 1.5 W/cm2. Histologic and immunohistochemical staining showed that increasing LIFU intensities were associated with increasing amounts of mucosal thickening, inflammatory cell infiltration, and extravasation of fibrin-
ogen. Our results showed that LIFU at 1.0W/cm2 with a duty cycle of 20% for 30 s was the maximum setting that could be used without causing histologically evident damage in the mouse middle ear mucosa. In addition, fibrinogen staining was seen around the vessels and middle ear mucosa under this condition. We coupled these LIFU effects with varying doses of ampicillin, which showed a dose-dependent antibacterial effect, as expected. The number of inflammatory cells and viable bacteria, and the levels of IL-1b and TNF-a in MEE were significantly lower in the LIFU(1) group treated with 10 mg/kg ampicillin than in the LIFU(2) group treated with the same ampicillin dose. Furthermore, the number of viable bacteria and levels of TNFa in MEE were significantly lower in the LIFU(1) group treated with 2 mg/kg ampicillin than in the LIFU(2) group treated with the same ampicillin dose. However, these outcome variables did not differ significantly between LIFU(1) and LIFU(2) groups that were treated with the higher ampicillin doses (50 mg/kg) or with no ampicillin (i.e., with PBS). A likely explanation for these findings is that an ampicillin dose of 50 mg/kg is so extreme that it achieves complete or sufficient tissue delivery, regardless of other factors such as LIFU. In addition, there was a trend toward improvement in the
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Fig. 8. The effect of LIFU on IL-1b and TNF-a levels in MEEs. (a) The differences in the concentration of IL-1b reached statistical significance in the groups treated with 10 mg/kg ampicillin (mean concentration of IL-1b 6 SD in LIFU[2] vs. LIFU(1), respectively; 236 6 174 vs. 42 6 70, p 5 0.003). (b) The concentration of TNF-a in MEEs were also significantly lower in the LIFU(1)/10 mg and 2 mg/kg ampicillin group than in the LIFU(2) group (mean concentration of TNF-a 6 SD in LIFU[2] vs. LIFU(1), respectively; 10 mg, 68 6 54 vs. 12 6 22, p 5 0.017; 2 mg, 90 6 48 vs. 45 6 37, p 5 0.048). LIFU 5 low-intensity focused ultrasound; PBS 5 phosphate-buffered saline.
severity of AOM in the LIFU(1) groups compared with the LIFU(2) groups; however, the difference in AOM severity scores did not achieve statistical significance between the two groups at any dose of ampicillin. It is known that middle ear findings used to determine the severity of AOM are affected by endotoxin, which is a component of the outer membrane of all gramnegative bacteria and consists of lipo-oligosaccharide complexed with outer membrane proteins of NTHi. It has been reported that endotoxin is present in MEE of up to 80% of patients who suffer from chronic otitis media with effusion and that 67% of culture-negative MEE samples contain endotoxin (DeMaria et al. 1984). Endotoxin is also a potent inducer of inflammation and a modulator of immune responses in the middle ear (Willett et al. 1998). Moreover, administration of endotoxin alone into the middle ear induces otitis media with effusion in animals (DeMaria and Murwin 1997; Watanabe et al. 2001). These results indicate that the
severity of AOM as determined by MEE findings might be affected by not only viable bacteria but also bacterial components such as endotoxin and outer membrane proteins. As these results, the difference between the two groups in the AOM severity score was not statistically significant in this study. In previous studies, extravasation of fibrinogen was used as a sensitive marker to show vessel disruption in the cochlea and submandibular gland (Ichimiya et al. 1999; Shuto et al. 2006). Shuto et al. (2006) reported that immunostaining after LIFU application showed the presence of extravascular fibrinogen in the submandibular gland, and that disruption of the vessel wall continued for at least 5 d after LIFU application. Our findings indicated that the application of LIFU to exposed mouse middle ear mucosa produced transient disruption of the vessels, allowing fibrinogen to diffuse from the vessels for several days. The molecular weight of murine fibrinogen (soluble dimer) is 340 kDa. It may be that drugs of
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Fig. 9. The effect of LIFU on IL-1b and TNF-a mRNA expression in inflammatory cells. The IL-1b mRNA (a) and TNFa mRNA (b) expression levels did not differ significantly between most LIFU(1) mice and the LIFU(2) mice that received the same ampicillin dose. However, both cytokine expression levels were slightly lower in the LIFU(1)/ 10 mg/kg ampicillin group than in the LIFU(2)/10 mg/kg ampicillin group. LIFU 5 low-intensity focused ultrasound; PBS 5 phosphate-buffered saline.
similar molecular weight can pass through the vessels to enter target organs; this has considerable clinical implications and can be particularly applicable to various antibiotic agents. The results from Yu et al. (2012) also support ours, and they reported that low-intensity ultrasound alone is not effective in killing bacteria, whereas the combination of low intensity ultrasound and antibiotics is promising. He et al. (2011) also reported the effectiveness of LIFU with microbubbles on biofilms of Streptococcus epidermidis. Biofilm densities and the viable counts of bacteria recovered from the biofilm were significantly decreased in biofilms treated with vancomycin and microbubbles combined with 1.0 W/ cm2 LIFU. Ours is the first report in which LIFU with antibiotic treatment is shown to be effective in enhancing bacterial clearance and reducing inflammatory responses in the middle ear with AOM. LIFU application along with antibiotics may be an effective strategy for the treatment of AOM, although further study is
needed to estimate the amount of acoustic energy being delivered to the middle ear to confirm the effect of LIFU and also to protect the inner ear against damage to move closer to establishing LIFU clinical trials in human patients with AOM. CONCLUSION Our results show that in vivo LIFU can disrupt vessels and increase vessel permeability in the mouse middle ear mucosa without causing tissue damage. The first part of this study showed the maximum LIFU parameters that would not cause histologic damage in the murine middle ear mucosa: 1.0 W/cm2 with a duty cycle of 20% for 30 s. When ampicillin was given for 3 d at intraperitoneal doses of 2 and 10 mg/kg, thenumber of inflammatory cells, viable bacteria counts, and levels of IL-1b and TNF-a in MEEs were significantly lower when LIFU was administered to the
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ear than when it was not. LIFU application along with antibiotics may be an effective strategy for treatment of AOM. Acknowledgments—This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan (22591885). We thank A. Iwamoto in our department for assistance.
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