DMSO is an amphipathic molecule with a polar domain and two apolar methyl groups making it soluble in both aqueous and organic media. It is one of the most common solvents for dissolving hydrophobic compounds employed in vivo
or in vitro
studies. DMSO has frequently been used in studies of the inner ear; however, its potential side effects are unknown and have been largely overlooked in previous studies (Basile et al., 1996
; Guitton et al., 2003
; Harris et al., 2005
; Lee et al., 2002; So et al., 2005
). Our in vitro
results obtained with postnatal cochlear cultures showed little or no evidence of hair cell damage following 24 h treatment with 0.25% DMSO or less. These results are consistent with two previous reports that used 0.1% DMSO and found no negative side effects (Lee et al., 2001; Matsui et al., 2002
). A 0.5% DMSO concentration caused a slight increase in IHC (). However, once the concentration reached 0.75% there were clear signs of damage to IHC over most of the cochlea as well as damage to OHC in the base of the cochlea. DMSO concentrations of 1% have been used with embryonic day 13 and 18 mouse organ cultures, but there was no report of damage in these cultures (Mueller et al., 2002
). The absence of DMSO-induced damage could conceivably due to the addition of heparin, which has been reported to protect cells from damage (Han et al., 2005
; Slofstra et al., 2005
). Another factor that could play a role is the age of the cochlear cultures. Hair cells in embryonic day 13 and 18 cultures are much less mature that those in our postnatal day 2−3 cultures.
DMSO concentrations of 2−3% caused mild to moderate stereocilia damage and disarray and some swelling of the hair cell body. DMSO concentrations of 5−6% lead to the loss of stereocilia bundles, depletion of actin within the cuticular plate and significant swelling of the hair cells throughout cochlea. The pattern of hair cell loss induced by DMSO progressed from base to apex consistent with most other ototoxic drugs (Ding et al., 2002
; Kamimura et al., 1999
). In cochlear cultures, ototoxic drugs typically damage the OHC first followed by IHC (Ding et al., 2002
; Zhang et al., 2003
). However, a major difference in the damage pattern was that DMSO caused greater loss of IHC than OHC. This was clearly apparent at a concentration of 0.75% where IHC loss was evident over the entire length of the cochlea, yet OHC loss was present only in the extreme base.
Even though DMSO induced significant hair cell loss, the nearby Hensen cells had normal nuclei and were negative for TUNEL, caspase-3, caspase-8 and caspase-9 (). These results clearly indicate that hair cells are more vulnerable to DMSO than the supporting cells and that IHC are more susceptible to DMSO damage than OHC. The reasons for these cellular differences in DMSO cytotoxicity are unclear. Other cell types appear to be more resistant to the cytotoxic effects of DMSO than hair cells. For example, DMSO concentration of 10% had no cytotoxic effects on Caco2/TC7 cells (Da Violante et al., 2002
Oncosis, sometimes referred to as necrosis, is a form of cell death that is characterized by enlarged cellular organelles and swelling of the cell's membrane, which can cause the plasma membrane to rupture and release its contents into the extracellular environment (Bohne et al., 2007
; Jaeschke et al., 2003; Majno et al., 1995). High doses of DMSO induced significant swelling of the hair cells suggesting that hair cells might be dying by oncosis. However, our morphological and biochemical assessment indicated that most hair cells in the DMSO treated cultures had shrunken and condensed nuclei, and stained positive for TUNEL, caspase-3, caspase-8, and caspase-9. These later results suggest that DMSO induced hair cell death was occurring predominantly by apoptosis (Jaeschke et al., 2003). Our caspase-9 results are in line with the apoptotic cell death pattern seen in murine lymphoma cells where DMSO caused a collapse of the mitochondrial membrane potential, release of cytochrome c from the mitochondria followed by activation of caspase-9 and caspase-3 (Liu et al., 2001
). However, unlike cochlear hair cells, DMSO failed to activate the extrinsic caspase-8 pathway in lymphoma cells.
In summary, our results indicate that 24 h treatment with DMSO concentrations of 0.25% or less caused little or no damage to postnatal hair cells in cochlear organotypic cultures. DMSO is known to be a scavenger of the hydroxyl radical and it is possible that low concentrations of DMSO will be protective in cochlear cultures (Repine et al., 1981
). However, this should be regarded as speculative at this time since there is no direct evidence for a protective effect in our data. However, our results clearly indicate that DMSO concentrations of 0.5% or higher are toxic to both IHC and OHC in postnatal rat cochlear cultures. DMSO-induced hair cell damage progressed from base to apex; however, unlike other ototoxic compounds IHC were more vulnerable than OHC. Thus, culture media with DMSO concentrations equal to or greater than 25% are likely to be damaging to postnatal cochlear cultures.
DMSO is sometimes used to dissolve drugs that are applied to the inner ear via the middle ear or round window (Harris et al., 2005
; Liu et al., 2006
); however, it is unclear if DMSO is toxic to adult hair cells in vivo
. It is conceivable that adult hair cells are more resistant to the toxic effects of DMSO. However, given that high concentrations of DMSO are toxic in vitro
, future studies should be carried out to assess the toxicity of DMSO in vivo.
While in vivo
studies are important clinically, determining the dose-response relationship for DMSO is challenging. It is technically difficult to control the volume of DMSO that is applied to and remains on the round window membrane. Moreover, it is difficult to know how long it takes DMSO to pass through the round window; how long it takes to diffuse from the base to the apex of the cochlea and what the actual concentration is at any location and point in time. Since the volume of fluid within the cochlea is relatively large compared to the volume of DMSO that can be applied to the round window, the actual concentration of DMSO reaching the hair cells in vivo
may be quite low. An alternative method for assessing the vulnerability of IHC and OHC to DMSO in vivo
would be to deliver DMSO into scala tympani using an osmotic pump with a specified flow rate and concentration. This method would seem to offer the greatest precision for establishing the dose-response toxicity of DMSO in vivo
. With the increasing likelihood that therapeutic agents will be delivered directly to the cochlear, this clinically relevant question merits further study.