Eur Arch Otorhinolaryngol DOI 10.1007/s00405-014-2957-y
Review article
Effect of intratympanic dimethyl sulphoxide (DMSO) in an in vivo model of cisplatin‑related ototoxicity A. Roldán‑Fidalgo · A. Trinidad · A. Rodríguez‑Valiente · J. R. García‑Berrocal · I. Millán · M. J. Coronado · R. Ramírez‑Camacho
Received: 23 September 2013 / Accepted: 17 February 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Although dimethyl sulfoxide (DMSO) is one of the most common solvents employed in otoprotection studies, its effect on the inner ear remains unknown. Only a few in vitro studies have addressed the effect of DMSO in cochlear cells. Up to the date, no in vivo functional studies have been reported. To determine the effect of intratympanic DMSO application in the inner ear, and to evaluate its effect in combination with cisplatin in Wistar rats, twelve Wistar rats were randomly assigned into two groups. Group A received intratympanic 1 % DMSO in both ears. Group B received intraperitoneal cisplatin (10 mg/kg) and intratympanic 0.5 % DMSO in the right ear and saline solution in the left ear. Functional changes were evaluated with Auditory Steady-State Responses before and 5 days after the procedure. Morphological changes were studied by means of confocal laser scanning microscopy following the removal of the temporal bones and cochlear dissection. Hearing threshold levels in group A did not show any statistically significant changes after the treatment. In group B, significant differences between pre- and post-treatment were found, with no statistically significant variations between right (DMSO) and left ear (saline solution). We A. Roldán‑Fidalgo (*) · A. Trinidad · A. Rodríguez‑Valiente · J. R. García‑Berrocal · R. Ramírez‑Camacho Department of Otorhinolaryngology, University Hospital Puerta de Hierro-Majadahonda, C/Manuel de Falla 1, Majadahonda, 28222 Madrid, Spain e-mail: [email protected] I. Millán Department of Statistics, University Hospital Puerta de Hierro-Majadahonda, Madrid, Spain M. J. Coronado Confocal Scanning Laser Microscopy Unit, University Hospital Puerta de Hierro-Majadahonda, Madrid, Spain
suggest that DMSO could be safely used to dissolve hydrophobic compounds in otoprotection studies without interfering with the cochlear damage produced by cisplatin. Keywords Cisplatin · Dimethyl sulfoxide · Ototoxicity · Confocal laser scanning microscopy · Auditory steady-state responses
Introduction Cisplatin is a commonly prescribed platinum-based drug used to treat different types of solid tumors with a wellknown ototoxic effect. There is a great interest in developing effective strategies to protect the inner ear without affecting the antitumoral activity of cisplatin. Intratympanic administration has proved that many substances are able to cross the round window membrane [1]. It has been observed that intratympanic administration of some drugs such as steroids achieves higher concentrations in the inner ear than either oral or intravenous administration [2]. Many research groups evaluated the intratympanic application of protective agents to prevent cisplatin ototoxicity. Most of these agents are usually dissolved in dimethyl sulfoxide (DMSO). The effect of DMSO has been studied in some types of in vitro and in vivo cancers [3–5], but the effect of this compound in the inner ear has not yet been defined. Dimethyl sulphoxide is a polar amphipathic solvent, with anti-inflammatory and anti-oxidant properties [6], often used to dissolve hydrophobic substances at in vivo and in vitro studies. It facilitates the diffusion of different compounds inside the cell when it is applied to isolated hair cell cultures [7–10], increases the solubility of these substances through the round window membrane and blocks the rise of intracellular calcium induced by different agents
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[10–16]. DMSO has been successfully used in the treatment of gastrointestinal [17, 18], rheumatic [19], urinary [20], pulmonary [21], dermatological [22, 23], and brain disorders [24, 25]. Central nervous system studies have shown that DMSO suppresses the α-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) and N-metild-aspartato (NMDA) receptors and attenuates the calcium responses to glutamate [15], preventing a proapoptotic cascade activation [26], which is thought to be a key mechanism of glutamate excitotoxicity. Although DMSO is one of the most commonly employed solvents in mammalians otoprotection studies, only two have concerned about the potential ototoxic effect of this molecule [3, 10], whereas a great number of articles did not mention this potential side effect [27–33]. Melki et al. and Momin et al. reported significant hearing preservation by DMSO in endolymphatic hydrop models when it was administrated parenterally [4, 14]. In zebrafish models [3, 10, 34–41], most of the compounds used in cisplatin ototoxicity studies were dissolved in DMSO. The aims of this study were to determine the effect of intratympanic DMSO on the inner ear function and morphology, and to evaluate its effect when it is combined with cisplatin.
Methods Animals Female Wistar rats weighing 230–275 g were used in the present study. Animals were breaded and handled at the animal facility of the Hospital Puerta de Hierro in temperature controlled rooms, with light–dark cycles, and with free access to food and water. Experimental procedures were performed in the experimental operating rooms. The study was carried out in accordance with the guidelines for research involving animals (Spanish Animal Care and Use Committee, Spanish law 32/2007 and European regulation 86/609/CEE), and was approved by the Animal Welfare Ethics Committee of the Foundation for Biomedical Research of the Hospital Puerta de Hierro (CEBA 013/2012). Otoscopy was performed using an operating microscope to exclude animals with outer or middle ear infections. Twelve Wistar rats were randomly assigned into two groups. Group A (n = 5) received intratympanic 1 % DMSO (0.03 ml) in both ears. Group B (n = 7) received intratympanic 0.5 % DMSO (0.03 ml) in the right ear, and saline solution (0.03 ml) in the left ear, in combination with intraperitoneal cisplatin (10 mg/kg). DMSO (Sigma Aldrich) was diluted in phosphate buffered saline (PBS).
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Eur Arch Otorhinolaryngol
Experimental procedure Animals were anesthetized with intraperitoneal ketamine (100 mg/kg) and diazepam (0.1 mg/kg). An initial auditory steady-state responses (ASSR) test was performed on all animals. An insert earphone (Etymotic Research ER-2) was placed directly into the external auditory canal. Subcutaneous electrodes were placed over the vertex (active) and in the pinna of each ear (reference). Ground electrodes were placed over the neck muscles. ASSR were recorded using an evoked potential averaging system (Intelligent Hearing System Smart-EP, FL, USA) in an electrically shielded, double-walled, sound-treated booth in response to 100 ms clicks or tone burst at 8, 12, 16, 20, 24 and 32 kHz with 10 ms plateau and 1 ms rise/fall time. Intensity was expressed in decibels sound pressure level (dB SPL) peak equivalent. Intensity series were recorded, and an ASSR threshold was defined by the lowest intensity able to induce a replicable visual detectable response. Following the ASSR measurements, 0.03 ml of DMSO was injected transtympanically using a spinal needle (BD Whitecare 27G). After each injection the anesthetized animals remained in lateral decubitus for 30 min to maximize the solution’s contact time with the round window membrane and to prevent its leakage into the pharynx through the eustachian tube. This procedure was equally performed for both groups. In group B (DMSO + cisplatin), after intratympanic DMSO administration in the right ear and saline solution in the left ear, an intraperitoneal slow infusion of cisplatin (Pharma-Pfizer, 10 mg/kg) was carried out for 30 min. After the cisplatin infusion, animals were housed in individual cages with ad libitum water and food. Auditory steady-state response was tested in each animal 5 days after the procedure. Immediately after completion of follow-up ASSR, anesthetized rats were euthanized by decapitation, and temporal bones were removed for histological study. Statistical analysis of the results was performed once the sample size had five animals in group A and seven animals in group B. The analyses showed that the sample size was large enough to achieve statistically significant results. According to Spanish and European legislation regarding the use of the least number of animals necessary to obtain the required results in experiments with animals, we decided not to add more subjects to the study. Histology The cochlear portion of the temporal bone was isolated. Stapes were removed and round window membrane was carefully incised, to perfuse paraformaldehyde through the round window and rinse the endolymphatic
Eur Arch Otorhinolaryngol
and perilymphatic spaces. The whole piece was fixed in paraformaldehyde for 24 h and decalcified in 1 % ethylenediaminetetraacetic acid (EDTA) at room temperature (with daily changes) for 10–12 days. Once the bone was completely decalcified, cochleae were dissected in PBS medium, and cochlear surface extracts were laid for immunohistochemistry and confocal microscopy analysis. All extracts were stained with To-Pro (1/1000 Invitrogene) to evaluate nuclei, and phalloidin (1/25 Sigma) to evaluate the morphological changes for actin immunostaining. Specimens were then examined under a confocal laser scanning microscope (Leyca Microsystems, Wetzlar, Germany) to determine the DMSO effects for each group. Hair cell damage was observed in the basal, medial and apical extracts. The total number of outer hair cells (OHC) and its loss (considered by the presence of empty spaces instead of OHC) were reported in each extract. Total OHC loss average was performed for each group of the study. Cell counts were performed at the time of imaging by sequentially viewing the image slices.
Fig. 1 Mean and standard deviation values of hearing thresholds before and after the treatment in group A (right and left ear)
Statistical analysis To evaluate the potential ototoxicity of DMSO alone or in combination with cisplatin, one-factorial and two-factorial analysis of variance (ANOVA) using the SPSS-15 software were performed. Paired tests were done comparing both ears, and a different treatment was evaluated to identify any statistically significant effect (p
Review article
Effect of intratympanic dimethyl sulphoxide (DMSO) in an in vivo model of cisplatin‑related ototoxicity A. Roldán‑Fidalgo · A. Trinidad · A. Rodríguez‑Valiente · J. R. García‑Berrocal · I. Millán · M. J. Coronado · R. Ramírez‑Camacho
Received: 23 September 2013 / Accepted: 17 February 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Although dimethyl sulfoxide (DMSO) is one of the most common solvents employed in otoprotection studies, its effect on the inner ear remains unknown. Only a few in vitro studies have addressed the effect of DMSO in cochlear cells. Up to the date, no in vivo functional studies have been reported. To determine the effect of intratympanic DMSO application in the inner ear, and to evaluate its effect in combination with cisplatin in Wistar rats, twelve Wistar rats were randomly assigned into two groups. Group A received intratympanic 1 % DMSO in both ears. Group B received intraperitoneal cisplatin (10 mg/kg) and intratympanic 0.5 % DMSO in the right ear and saline solution in the left ear. Functional changes were evaluated with Auditory Steady-State Responses before and 5 days after the procedure. Morphological changes were studied by means of confocal laser scanning microscopy following the removal of the temporal bones and cochlear dissection. Hearing threshold levels in group A did not show any statistically significant changes after the treatment. In group B, significant differences between pre- and post-treatment were found, with no statistically significant variations between right (DMSO) and left ear (saline solution). We A. Roldán‑Fidalgo (*) · A. Trinidad · A. Rodríguez‑Valiente · J. R. García‑Berrocal · R. Ramírez‑Camacho Department of Otorhinolaryngology, University Hospital Puerta de Hierro-Majadahonda, C/Manuel de Falla 1, Majadahonda, 28222 Madrid, Spain e-mail: [email protected] I. Millán Department of Statistics, University Hospital Puerta de Hierro-Majadahonda, Madrid, Spain M. J. Coronado Confocal Scanning Laser Microscopy Unit, University Hospital Puerta de Hierro-Majadahonda, Madrid, Spain
suggest that DMSO could be safely used to dissolve hydrophobic compounds in otoprotection studies without interfering with the cochlear damage produced by cisplatin. Keywords Cisplatin · Dimethyl sulfoxide · Ototoxicity · Confocal laser scanning microscopy · Auditory steady-state responses
Introduction Cisplatin is a commonly prescribed platinum-based drug used to treat different types of solid tumors with a wellknown ototoxic effect. There is a great interest in developing effective strategies to protect the inner ear without affecting the antitumoral activity of cisplatin. Intratympanic administration has proved that many substances are able to cross the round window membrane [1]. It has been observed that intratympanic administration of some drugs such as steroids achieves higher concentrations in the inner ear than either oral or intravenous administration [2]. Many research groups evaluated the intratympanic application of protective agents to prevent cisplatin ototoxicity. Most of these agents are usually dissolved in dimethyl sulfoxide (DMSO). The effect of DMSO has been studied in some types of in vitro and in vivo cancers [3–5], but the effect of this compound in the inner ear has not yet been defined. Dimethyl sulphoxide is a polar amphipathic solvent, with anti-inflammatory and anti-oxidant properties [6], often used to dissolve hydrophobic substances at in vivo and in vitro studies. It facilitates the diffusion of different compounds inside the cell when it is applied to isolated hair cell cultures [7–10], increases the solubility of these substances through the round window membrane and blocks the rise of intracellular calcium induced by different agents
13
[10–16]. DMSO has been successfully used in the treatment of gastrointestinal [17, 18], rheumatic [19], urinary [20], pulmonary [21], dermatological [22, 23], and brain disorders [24, 25]. Central nervous system studies have shown that DMSO suppresses the α-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) and N-metild-aspartato (NMDA) receptors and attenuates the calcium responses to glutamate [15], preventing a proapoptotic cascade activation [26], which is thought to be a key mechanism of glutamate excitotoxicity. Although DMSO is one of the most commonly employed solvents in mammalians otoprotection studies, only two have concerned about the potential ototoxic effect of this molecule [3, 10], whereas a great number of articles did not mention this potential side effect [27–33]. Melki et al. and Momin et al. reported significant hearing preservation by DMSO in endolymphatic hydrop models when it was administrated parenterally [4, 14]. In zebrafish models [3, 10, 34–41], most of the compounds used in cisplatin ototoxicity studies were dissolved in DMSO. The aims of this study were to determine the effect of intratympanic DMSO on the inner ear function and morphology, and to evaluate its effect when it is combined with cisplatin.
Methods Animals Female Wistar rats weighing 230–275 g were used in the present study. Animals were breaded and handled at the animal facility of the Hospital Puerta de Hierro in temperature controlled rooms, with light–dark cycles, and with free access to food and water. Experimental procedures were performed in the experimental operating rooms. The study was carried out in accordance with the guidelines for research involving animals (Spanish Animal Care and Use Committee, Spanish law 32/2007 and European regulation 86/609/CEE), and was approved by the Animal Welfare Ethics Committee of the Foundation for Biomedical Research of the Hospital Puerta de Hierro (CEBA 013/2012). Otoscopy was performed using an operating microscope to exclude animals with outer or middle ear infections. Twelve Wistar rats were randomly assigned into two groups. Group A (n = 5) received intratympanic 1 % DMSO (0.03 ml) in both ears. Group B (n = 7) received intratympanic 0.5 % DMSO (0.03 ml) in the right ear, and saline solution (0.03 ml) in the left ear, in combination with intraperitoneal cisplatin (10 mg/kg). DMSO (Sigma Aldrich) was diluted in phosphate buffered saline (PBS).
13
Eur Arch Otorhinolaryngol
Experimental procedure Animals were anesthetized with intraperitoneal ketamine (100 mg/kg) and diazepam (0.1 mg/kg). An initial auditory steady-state responses (ASSR) test was performed on all animals. An insert earphone (Etymotic Research ER-2) was placed directly into the external auditory canal. Subcutaneous electrodes were placed over the vertex (active) and in the pinna of each ear (reference). Ground electrodes were placed over the neck muscles. ASSR were recorded using an evoked potential averaging system (Intelligent Hearing System Smart-EP, FL, USA) in an electrically shielded, double-walled, sound-treated booth in response to 100 ms clicks or tone burst at 8, 12, 16, 20, 24 and 32 kHz with 10 ms plateau and 1 ms rise/fall time. Intensity was expressed in decibels sound pressure level (dB SPL) peak equivalent. Intensity series were recorded, and an ASSR threshold was defined by the lowest intensity able to induce a replicable visual detectable response. Following the ASSR measurements, 0.03 ml of DMSO was injected transtympanically using a spinal needle (BD Whitecare 27G). After each injection the anesthetized animals remained in lateral decubitus for 30 min to maximize the solution’s contact time with the round window membrane and to prevent its leakage into the pharynx through the eustachian tube. This procedure was equally performed for both groups. In group B (DMSO + cisplatin), after intratympanic DMSO administration in the right ear and saline solution in the left ear, an intraperitoneal slow infusion of cisplatin (Pharma-Pfizer, 10 mg/kg) was carried out for 30 min. After the cisplatin infusion, animals were housed in individual cages with ad libitum water and food. Auditory steady-state response was tested in each animal 5 days after the procedure. Immediately after completion of follow-up ASSR, anesthetized rats were euthanized by decapitation, and temporal bones were removed for histological study. Statistical analysis of the results was performed once the sample size had five animals in group A and seven animals in group B. The analyses showed that the sample size was large enough to achieve statistically significant results. According to Spanish and European legislation regarding the use of the least number of animals necessary to obtain the required results in experiments with animals, we decided not to add more subjects to the study. Histology The cochlear portion of the temporal bone was isolated. Stapes were removed and round window membrane was carefully incised, to perfuse paraformaldehyde through the round window and rinse the endolymphatic
Eur Arch Otorhinolaryngol
and perilymphatic spaces. The whole piece was fixed in paraformaldehyde for 24 h and decalcified in 1 % ethylenediaminetetraacetic acid (EDTA) at room temperature (with daily changes) for 10–12 days. Once the bone was completely decalcified, cochleae were dissected in PBS medium, and cochlear surface extracts were laid for immunohistochemistry and confocal microscopy analysis. All extracts were stained with To-Pro (1/1000 Invitrogene) to evaluate nuclei, and phalloidin (1/25 Sigma) to evaluate the morphological changes for actin immunostaining. Specimens were then examined under a confocal laser scanning microscope (Leyca Microsystems, Wetzlar, Germany) to determine the DMSO effects for each group. Hair cell damage was observed in the basal, medial and apical extracts. The total number of outer hair cells (OHC) and its loss (considered by the presence of empty spaces instead of OHC) were reported in each extract. Total OHC loss average was performed for each group of the study. Cell counts were performed at the time of imaging by sequentially viewing the image slices.
Fig. 1 Mean and standard deviation values of hearing thresholds before and after the treatment in group A (right and left ear)
Statistical analysis To evaluate the potential ototoxicity of DMSO alone or in combination with cisplatin, one-factorial and two-factorial analysis of variance (ANOVA) using the SPSS-15 software were performed. Paired tests were done comparing both ears, and a different treatment was evaluated to identify any statistically significant effect (p
