The aims of this study were to investigate the effects of intratympanic injections of isosorbide on vestibular function in animal models of endolymphatic hydrops and to find a new treatment option for the acute onset of vertigo in Ménière disease (MD).
Seventy male guinea pigs received intratympanic injection of isosorbide (IT-ISB). The animals were divided into three study groups: control, a chronic hydrops model, and an acute hydrops model. Intracochlear drug concentrations were measured using high-performance liquid chromatography. Vestibular function was analyzed using an animal rotator test with bidirectional sinusoidal harmonic acceleration before and after IT-ISB administration. Histological changes were also investigated.
ISB successfully permeated the perilymph through the round window membrane (RWM) at all three concentrations (25%, 50%, and 100%). In the chronic hydrops model, while IT-ISB histologically induced a reduction of endolymphatic hydrops, vestibular function was unchanged. In the acute hydrops model, no endolymphatic hydrops was histologically observed, and vestibular symmetry was also preserved after IT-ISB.
ISB passed through the RWM into the perilymphatic space even at lower concentrations. IT-ISB histologically reduced hydrops in the chronic model and preserved symmetrical vestibular function in the acute model. IT-ISB could be a treatment candidate for acute attacks of vertigo in MD.
The treatment of Ménière disease (MD) has progressed slowly, although it has been the focus of many studies, and no treatment of choice has yet been established to inhibit the progression of the disease [
Osmotic diuretics have recently been used for the treatment of MD on the basis of studies indicating that these agents can reduce endolymphatic hydrops, based on histological, biochemical, and electrophysiological evidence of efficacy [
Although the oral administration of ISB (PO-ISB) improved endolymphatic hydrops in an animal model [
To study the effect of IT-ISB on endolymphatic hydrops, it is critical to use an appropriate animal hydrops model. Although the traditional chronic model using endolymphatic sac surgery [
A total of 78 male guinea pigs with Preyer’s reflex (Hartley guinea pigs, 350–400 g) were used. We divided the animals into three study groups as follows (
The group 1 was divided into nine subgroups according to collection time of perilymph (0, 3, and 6 hours) and drug concentration (25%, 50%, and 100%) as below. Considering the loss due to the invasive perilymph sampling, seven animals were assigned to each subgroups. For anesthesia induction, xylazine (5 mg/kg, IM) and ketamine (35 mg/kg, IM) were administered. This study was approved by the University Animal Care and Use Committee (IACUC 150617-365).
As described in the previous study [
For IT-ISB, the bulla was opened with cutting burrs to expose the round window via the retro-auricular approach as in a previous study [
Unexpected spontaneous nystagmus, beating to the non-perfusion ear side, was observed (
After incision of the RWM, 2-μL perilymph was gently harvested with an ultra-fine pipette tip (Multimax 0.1–10 μL long-reach tip; Sorenson Bioscience, Salt Lake City, UT, USA) attached to a micropipette to avoid damage to the basilar membrane [
For chromatographic separation of ISB and analysis of its intracochlear concentrations, high-performance liquid chromatography coupled with refractive index detection (HPLC-RI) was used. Each sample was diluted with 28 μL of distilled water and filtered through a regenerated cellulose filter (0.2-μm; Sartorius Co., Goettingen, Germany). The Acme 9000 HPLC system was equipped with RI750F RID (YL Instrument Co., Anyang, Korea) and an Aminex HPX-87H Column (Bio-Rad, Hercules, CA, USA). A mobile phase of 0.005 M sulfuric acid and a flow rate of 0.6 mL/min were applied. Data were acquired and analyzed with the Autochro-3000 chromatography processing program (YL Instrument Co.). To avoid error from blood contamination during sampling, the glucose concentration was simultaneously analyzed. As the previously reported normal glucose level in the perilymph [
To evaluate vestibular function, sinusoidal harmonic acceleration (SHA) with a bidirectional 60° rotation at a frequency of 0.2 Hz was conducted using a customized animal rotator after attaching a fluorescent marker on each pupil under topical anesthesia as in a previously reported study [
After fixation in 4% formaldehyde solution for 2 days, decalcification with ethylenediamine tetra-acetic acid was performed for approximately 4 weeks, followed by dehydration and embedding in paraffin. Blocks were sectioned parallel to the modiolar axis at a 6-mm thickness and stained with hematoxylin and eosin. Changes of Reissner’s membrane and endolymphatic space were observed under light microscopy.
ISB successfully permeated into the perilymph through the RWM after injection at all three concentrations (25%, 50%, and 100%). The concentrations in the perilymph were 59.77±25.79 mM immediately (n=5), 19.28±4.02 mM at 3 hours (n=7), and 13.56± 4.38 mM at 6 hours (n=4) after 25% IT-ISB; 213.13±106.65 (n=4), 152.42±24.33 (n=5), and 40.72±22.87 mM (n=7), respectively, after 50% IT-ISB; and 245.47±112.84 (n=4), 149.80± 65.39 (n=4), and 66.81±20.41 mM (n=4), respectively, after 100% IT-ISB. The intracochlear concentration of 50% IT-ISB was higher than that of 25% IT-ISB at 0, 3, and 6 hours (
After 50% IT-ISB, unexpected spontaneous nystagmus was observed for 6 to 9 hours in all animals. By contrast, no spontaneous nystagmus was observed after 25% IT-ISB, and the symmetry score (4.96%±4.68%) was within the normal range (range, 0.47%–11.22%).
At 16 weeks after ES ablation, symmetrical response was observed in the bidirectional SHA tests (2.94%±1.66%; range, 1.26%–4.89%) (
Before VP injection, a symmetrical response was observed in the bidirectional SHA tests (symmetry score, 6.50%±7.60%). However, approximately 1 hour after the VP injection, the symmetry score increased to 25.23%±9.84% in the bidirectional SHA tests (
After 25% IT-ISB along with VP, the vestibular symmetry was unchanged (10.18%±4.26% vs. 10.51%±8.01%,
Experimental [
Intratympanic drug injections, including dexamethasone and gentamicin therapy, have been widely used for the treatment of MD [
To study the effects of IT-ISB, we first used the traditional animal model created by surgical ablation of the endolymphatic sac (group 2). Histological decompression of endolymphatic hydrops was observed after IT-ISB. By contrast, we could not find a significant change in vestibular function. We assume that this finding resulted from a pre-existing deterioration of vestibular function and subsequent vestibular compensation in this model. As this model only represents the chronic stage of hydrops, the so-called burnout stage [
Next, to investigate the effect of IT-ISB on the acute aggravation of hydrops, we used the new vasopressin-induced hydrops model (group 3). In this model, a vasopressin injection is used to induce hydrops aggravation by water shift from the perilymph to the endolymph after surgical ablation of the endolymphatic sac [
There are several limitations of this study. First, the distribution of the animal numbers was substantially unbalanced. Since no previous study investigated changes in intracochlear concentrations over time after IT-ISB, we first had to determine the optimal concentration and test for possible ototoxicity in normal animals before starting IT-ISB in the animal model groups. For this reason, we had to use more animals in the normal animal group than in the animal model groups. Based on the results, it was possible to assign five animals equally to each animal model. Second, we did not investigate the effect of IT-ISB on audiologic function. Since we used the trans-bullar approach and high-viscosity ISB for the perfusion, which can substantially affect hearing thresholds, it would have been difficult to measure hearing adequately. To evaluate hearing changes in animal models, it is necessary to conduct a further study using a minimally invasive technique and ISB at a lower viscosity. Third, the present hydrops model is not yet perfect for the investigation of MD. Furthermore, the reason for discrepancies between histologic hydrops and MD remains controversial. Individual differences in susceptibility and resistance to MD are also not well understood. Further research is needed to clarify the pathophysiology of MD. In addition, the statistical reliability of our findings might have been low due to a small sample size. A follow-up study is necessary with a larger sample size, as well as an a posteriori power analysis.
Even though we found that IT-ISB could be feasible for the treatment of MD, further research is needed to clarify certain points. First, high ISB concentrations can induce transient vestibular dysfunction. An intratympanic injection of 100% ISB was reported to provoke paralytic nystagmus in rabbits and cats, even though it diminished in 3 hours [
In conclusion, ISB passed through the RWM into the perilymphatic space even at lower concentrations. IT-ISB histologically reduced hydrops in the chronic model and preserved symmetrical vestibular function in the acute model. IT-ISB could be a treatment candidate for vertigo in MD.
■ No treatment of choice has been established for acute attacks of vertigo in Ménière disease.
■ Isosorbide passed through the round window membrane even at lower concentrations.
■ Intratympanic injection of isosorbide histologically reduced hydrops in the chronic model and preserved symmetrical vestibular function in the acute model.
■ Intratympanic injection of isosorbide could be a treatment option for acute attacks of vertigo in Ménière disease.
No potential conflict of interest relevant to this article was reported.
Conceptualization: MK. Data curation: SYY. Formal analysis: MK. Funding acquisition: MK. Methodology: MK, HH, SYY. Project administration: HJH. Visualization: HH. Writing–original draft: MK. Writing–review & editing: MK, HH, HJH.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2017R1D1A3B03030968). We wish to express our most sincere gratitude and appreciation to Prof. Akinobu Kakigi for his guidance and advice.
Supplementary materials can be found via
Unexpected spontaneous nystagmus, beating to the non-perfusion ear side, was observed after the intratympanic injection of isosorbide.
Experimental groups. Group 1, the control group, was injected with three different isosorbide concentrations (IT-ISB). Group 2, the chronic hydrops model, was treated with IT-ISB at 12–16 weeks after endolymphatic sac (ES) ablation surgery. Group 3, the acute hydrops model, was treated with (3a) desmopressin (VP) injected at 2–4 weeks after ES surgery for the induction of hydrops aggravation and (3b) IT-ISB along with VP. IT-ISB, intratympanic injection of isosorbide.
Intracochlear concentrations of isosorbide after injections at different concentrations. Whiskers show the standard deviations. IT-ISB, intratympanic injection of isosorbide. *
Intratympanic injection of isosorbide (IT-ISB) in the chronic hydrops model. (A) No significant change of symmetrical vestibular function was observed after IT-ISB. (B) Endolymphatic hydrops decreased after IT-ISB (arrowheads) (H&E, ×40).
Intratympanic injection of isosorbide (IT-ISB) in the acute hydrops model. (A) Acute vestibular dysfunction after the induction with desmopressin (VP) and preserved vestibular symmetry after IT-ISB along with VP. Whiskers show the standard deviations. *