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1.  Regeneration of mammalian cochlear and vestibular hair cells through Hes1/Hes5 modulation with siRNA 
Hearing research  2013;304:91-110.
The Notch pathway is a cell signaling pathway determining initial specification and subsequent cell fate in the inner ear. Previous studies have suggested that new hair cells (HCs) can be regenerated in the inner ear by manipulating the Notch pathway. In the present study, delivery of siRNA to Hes1 and Hes5 using a transfection reagent or siRNA to Hes1 encapsulated within poly(lactide-co-glycolide acid) (PLGA) nanoparticles increased HC numbers in non-toxin treated organotypic cultures of cochleae and maculae of postnatal day 3 mouse pups. An increase in HCs was also observed in cultured cochleae and maculae of mouse pups pre-conditioned with a HC toxin (4-hydroxy-2-nonenal or neomycin) and then treated with the various siRNA formulations. Treating cochleae with siRNA to Hes1 associated with a transfection reagent or siRNA to Hes1 delivered by PLGA nanoparticles decreased Hes1 mRNA and up-regulated Atoh1 mRNA expression allowing supporting cells (SCs) to acquire a HC fate. Experiments using cochleae and maculae of p27kip1/-GFP transgenic mouse pups demonstrated that newly generated HCs trans-differentiated from SCs. Furthermore, PLGA nanoparticles are non-toxic to inner ear tissue, readily taken up by cells within the tissue of interest, and present a synthetic delivery system that is a safe alternative to viral vectors. These results indicate that when delivered using a suitable vehicle, Hes siRNAs are potential therapeutic molecules that may have the capacity to regenerate new HCs in the inner ear and possibly restore human hearing and balance function.
PMCID: PMC4051161  PMID: 23850665
hair cell regeneration; inner ear; Notch pathway; siRNA; nanoparticle; mouse
3.  Antioxidants Reduce Cellular and Functional Changes Induced by Intense Noise in the Inner Ear and Cochlear Nucleus 
The present study marks the first evaluation of combined application of the antioxidant N-acetylcysteine (NAC) and the free radical spin trap reagent, disodium 2,4-disulfophenyl-N-tert-butylnitrone (HPN-07), as a therapeutic approach for noise-induced hearing loss (NIHL). Pharmacokinetic studies and C-14 tracer experiments demonstrated that both compounds achieve high blood levels within 30 min after i.p injection, with sustained levels of radiolabeled cysteine (released from NAC) in the cochlea, brainstem, and auditory cortex for up to 48 h. Rats exposed to 115 dB octave-band noise (10–20 kHz) for 1 h were treated with combined NAC/HPN-07 beginning 1 h after noise exposure and for two consecutive days. Auditory brainstem responses (ABR) showed that treatment substantially reduced the degree of threshold shift across all test frequencies (2–16 kHz), beginning at 24 h after noise exposure and continuing for up to 21 days. Reduced distortion product otoacoustic emission (DPOAE) level shifts were also detected at 7 and 21 days following noise exposure in treated animals. Noise-induced hair cell (HC) loss, which was localized to the basal half of the cochlea, was reduced in treated animals by 85 and 64 % in the outer and inner HC regions, respectively. Treatment also significantly reduced an increase in c-fos-positive neuronal cells in the cochlear nucleus following noise exposure. However, no detectable spiral ganglion neuron loss was observed after noise exposure. The results reported herein demonstrate that the NAC/HPN-07 combination is a promising pharmacological treatment of NIHL that reduces both temporary and permanent threshold shifts after intense noise exposure and acts to protect cochlear sensory cells, and potentially afferent neurites, from the damaging effects of acoustic trauma. In addition, the drugs were shown to reduce aberrant activation of neurons in the central auditory regions of the brain following noise exposure. It is likely that the protective mechanisms are related to preservation of structural components of the cochlea and blocking the activation of immediate early genes in the auditory centers of the brain.
PMCID: PMC4010594  PMID: 24497307
acoustic trauma; cochlea; hair cell; brainstem; HPN-07; N-acetylcysteine; antioxidant
4.  Reduced Formation of Oxidative Stress Biomarkers and Migration of Mononuclear Phagocytes in the Cochleae of Chinchilla after Antioxidant Treatment in Acute Acoustic Trauma 
Objective. Inhibition of inflammation and free radical formation in the cochlea may be involved in antioxidant treatment in acute acoustic trauma. Procedure. Chinchilla were exposed to 105 dB sound pressure level octave band noise for 6 hours. One group of chinchilla was treated with antioxidants after noise exposure. Auditory brainstem responses, outer hair cell counts, and immunohistochemical analyses of biomarkers in the cochlea were conducted. Results. The antioxidant treatment significantly reduced hearing threshold shifts, outer hair cell loss, numbers of CD45+ cells, as well as 4-hydroxy-2-nonenal and nitrotyrosine formation in the cochlea. Conclusion. Antioxidant treatment may provide protection to sensory cells by inhibiting formation of reactive oxygen and nitrogen products and migration of mononuclear phagocytes in the cochlea. The present study provides further evidence of effectiveness of antioxidant treatment in reducing permanent hearing loss.
PMCID: PMC3179894  PMID: 21961007
5.  Magnetic Targeted Delivery of Dexamethasone Acetate across the Round Window Membrane in Guinea Pigs 
Magnetically susceptible PLGA nanoparticles will effectively target the round window membrane (RWM) for delivery of dexamethasone-acetate (Dex-Ac) to the scala tympani.
Targeted delivery of therapeutics to specific tissues can be accomplished using different targeting mechanisms. One technology includes iron oxide nanoparticles, susceptible to external magnetic fields. If a nanocomposite composed of biocompatible polymer (PLGA), magnetite, and Dex-Ac can be pulled into and across the mammalian RWM, drug delivery can be enhanced.
In vitro targeting and release kinetics of PLGA-magnetite-Dex-Ac nanoparticles first were measured using a RWM model. Next, these optimized nanocomposites were targeted to the RWM by filling the niche in anesthetized guinea pigs. A permanent magnet was placed opposite the RWM for 1 hour. Cochlear soft tissues, perilymph, and RWM were harvested after euthanasia and steroid levels were measured using HPLC.
Membrane transport, in vitro, proved optimal targeting using a lower particle magnetite concentration (1 versus 5 or 10 mg/ml). In vivo targeted PLGA-magnetite-Dex-Ac particles had an average size of 482.8 ± 158 nm (DLS) and an average zeta potential −19.9 ± 3.3 mV. In 1 hour, there was significantly increased cochlear targeted delivery of Dex or Dex-Ac, compared with diffusion alone.
Superparamagnetic PLGA-magnetite-Dex-Ac nanoparticles under an external magnetic field (0.26 mT) for 1 hour significantly increased Dex-Ac delivery to the inner ear. The RWM was not completely permeated and also became loaded with nanocomposites, indicating that delivery to the cochlea would continue for weeks by PLGA degradation and passive diffusion.
PMCID: PMC3522431  PMID: 23187928
Dexamethasone acetate; Magnetic targeting; Poly(lactide-co-glycolide); Round window membrane; Sensorineural hearing loss
6.  Effects of Antioxidant Treatment on Blast-Induced Brain Injury 
PLoS ONE  2013;8(11):e80138.
Blast-induced traumatic brain injury has dramatically increased in combat troops in today’s military operations. We previously reported that antioxidant treatment can provide protection to the peripheral auditory end organ, the cochlea. In the present study, we examined biomarker expression in the brains of rats at different time points (3 hours to 21 days) after three successive 14 psi blast overpressure exposures to evaluate antioxidant treatment effects on blast-induced brain injury. Rats in the treatment groups received a combination of antioxidants (2,4-disulfonyl α-phenyl tertiary butyl nitrone and N-acetylcysteine) one hour after blast exposure and then twice a day for the following two days. The biomarkers examined included an oxidative stress marker (4-hydroxy-2-nonenal, 4-HNE), an immediate early gene (c-fos), a neural injury marker (glial fibrillary acidic protein, GFAP) and two axonal injury markers [amyloid beta (A4) precursor protein, APP, and 68 kDa neurofilament, NF-68]. The results demonstrate that blast exposure induced or up-regulated the following: 4-HNE production in the dorsal hippocampus commissure and the forceps major corpus callosum near the lateral ventricle; c-fos and GFAP expression in most regions of the brain, including the retrosplenial cortex, the hippocampus, the cochlear nucleus, and the inferior colliculus; and NF-68 and APP expression in the hippocampus, the auditory cortex, and the medial geniculate nucleus (MGN). Antioxidant treatment reduced the following: 4-HNE in the hippocampus and the forceps major corpus callosum, c-fos expression in the retrosplenial cortex, GFAP expression in the dorsal cochlear nucleus (DCN), and APP and NF-68 expression in the hippocampus, auditory cortex, and MGN. This preliminary study indicates that antioxidant treatment may provide therapeutic protection to the central auditory pathway (the DCN and MGN) and the non-auditory central nervous system (hippocampus and retrosplenial cortex), suggesting that these compounds have the potential to simultaneously treat blast-induced injuries in the brain and auditory system.
PMCID: PMC3818243  PMID: 24224042
7.  Nitrones as Therapeutics 
Free radical biology & medicine  2008;45(10):1361-1374.
Nitrones have the general chemical formula X-CH=NO-Y. They were first used to trap free radicals in chemical systems and then subsequently in biochemical systems. More recently several nitrones including PBN (α-phenyl-tert-butylnitrone) have been shown to have potent biological activity in many experimental animal models. Many diseases of aging including stroke, cancer development, Parkinson’s disease and Alzheimer’s disease are known to have enhanced levels of free radicals and oxidative stress. Some derivatives of PBN are significantly more potent than PBN and have undergone extensive commercial development in stroke. Recent research has shown that PBN-related nitrones also have anti-cancer activity in several experimental cancer models and have potential as therapeutics in some cancers. Also in recent observations nitrones have been shown to act synergistically in combination with antioxidants in the prevention of acute acoustic noise induced hearing loss. The mechanistic basis of the potent biological activity of PBN-related nitrones is not known. Even though PBN-related nitrones do decrease oxidative stress and oxidative damage, their potent biological anti-inflammatory activity and their ability to alter cellular signaling processes can not readily be explained by conventional notions of free radical trapping biochemistry. This review is focused on our observations and others where the use of selected nitrones as novel therapeutics have been evaluated in experimental models in the context of free radical biochemical and cellular processes considered important in pathologic conditions and age-related diseases.
PMCID: PMC2796547  PMID: 18793715
Nitrones; Stroke; Glioma; Hepatocellular Carcinoma; Hearing Loss; Acoustical Trauma
8.  Magnetic characterization of superparamagnetic nanoparticles pulled through model membranes 
To quantitatively compare in-vitro and in vivo membrane transport studies of targeted delivery, one needs characterization of the magnetically-induced mobility of superparamagnetic iron oxide nanoparticles (SPION). Flux densities, gradients, and nanoparticle properties were measured in order to quantify the magnetic force on the SPION in both an artificial cochlear round window membrane (RWM) model and the guinea pig RWM.
Three-dimensional maps were created for flux density and magnetic gradient produced by a 24-well casing of 4.1 kilo-Gauss neodymium-iron-boron (NdFeB) disc magnets. The casing was used to pull SPION through a three-layer cell culture RWM model. Similar maps were created for a 4 inch (10.16 cm) cube 48 MGOe NdFeB magnet used to pull polymeric-nanoparticles through the RWM of anesthetized guinea pigs. Other parameters needed to compute magnetic force were nanoparticle and polymer properties, including average radius, density, magnetic susceptibility, and volume fraction of magnetite.
A minimum force of 5.04 × 10-16 N was determined to adequately pull nanoparticles through the in-vitro model. For the guinea pig RWM, the magnetic force on the polymeric nanoparticles was 9.69 × 10-20 N. Electron microscopy confirmed the movement of the particles through both RWM models.
As prospective carriers of therapeutic substances, polymers containing superparamagnetic iron oxide nanoparticles were succesfully pulled through the live RWM. The force required to achieve in vivo transport was significantly lower than that required to pull nanoparticles through the in-vitro RWM model. Indeed very little force was required to accomplish measurable delivery of polymeric-SPION composite nanoparticles across the RWM, suggesting that therapeutic delivery to the inner ear by SPION is feasible.
PMCID: PMC1785374  PMID: 17204157
9.  The permeability of SPION over an artificial three-layer membrane is enhanced by external magnetic field 
Sensorineural hearing loss, a subset of all clinical hearing loss, may be correctable through the use of gene therapy. We are testing a delivery system of therapeutics through a 3 cell-layer round window membrane model (RWM model) that may provide an entry of drugs or genes to the inner ear. We designed an in vitro RWM model similar to the RWM (will be referred to throughout the paper as RWM model) to determine the feasibility of using superparamagnetic iron oxide (Fe3O4) nanoparticles (SPION) for targeted delivery of therapeutics to the inner ear.
The RWM model is a 3 cell-layer model with epithelial cells cultured on both sides of a small intestinal submucosal (SIS) matrix and fibroblasts seeded in between. Dextran encapsulated nanoparticle clusters 130 nm in diameter were pulled through the RWM model using permanent magnets with flux density 0.410 Tesla at the pole face. The SIS membranes were harvested at day 7 and then fixed in 4% paraformaldehyde. Transmission electron microscopy and fluorescence spectrophotometry were used to verify transepithelial transport of the SPION across the cell-culture model. Histological sections were examined for evidence of SPION toxicity, as well to generate a timeline of the position of the SPION at different times. SPION also were added to cells in culture to assess in vitro toxicity.
Transepithelial electrical resistance measurements confirmed epithelial confluence, as SPION crossed a membrane consisting of three co-cultured layers of cells, under the influence of a magnetic field. Micrographs showed SPION distributed throughout the membrane model, in between cell layers, and sometimes on the surface of cells. TEM verified that the SPION were pulled through the membrane into the culture well below. Fluorescence spectrophotometry quantified the number of SPION that went through the SIS membrane. SPION showed no toxicity to cells in culture.
A three-cell layer model of the human round window membrane has been constructed. SPION have been magnetically transported through this model, allowing quantitative evaluation of prospective targeted drug or gene delivery through the RWM. Putative in vivo carrier superparamagnetic nanoparticles may be evaluated using this model.
PMCID: PMC1475881  PMID: 16603066

Results 1-9 (9)