Biomedical subjects

Richard D Kopke

Publications and source records attributed to Richard D Kopke.

12 recordsLinked to original sources

PubMed2007

Magnetic characterization of superparamagnetic nanoparticles pulled through model membranes.

BACKGROUND: 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. METHODS: 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. RESULTS: A minimum force of 5.04 x 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 x 10-20 N. Electron microscopy confirmed the movement of the particles through both RWM models. CONCLUSION: 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.

Journal Article↗

PubMed2006

NAC for noise: from the bench top to the clinic.

Noise-induced hearing loss (NIHL) is an important etiology of deafness worldwide. Hearing conservation programs are in place and have reduced the prevalence of NIHL, but this disorder is still far too common. Occupational and recreational pursuits expose people to loud noise and ten million persons in the US have some degree of noise-induced hearing impairment. It is estimated that 50 million in the US and 600 million people worldwide are exposed to noise hazards occupationally. Noise deafness is still an important and frequent cause of battlefield injury in the US military. A mainstay of hearing conservation programs is personal mechanical hearing protection devices which are helpful but have inherent limitations. Research has shown that oxidative stress plays an important role in noise-induced cochlear injury resulting in the discovery that a number of antioxidant and cell death inhibiting compounds can ameliorate deafness associated with acoustic trauma. This article reviews one such compound, N-acetylcysteine (NAC), in terms of its efficacy in reducing hearing loss in a variety of animal models of acute acoustic trauma and hypothesizes what its therapeutic mechanisms of action might be based on the known actions of NAC. Early clinical trials with NAC are mentioned.

Acetylcysteine↗

PubMed2006

The permeability of SPION over an artificial three-layer membrane is enhanced by external magnetic field.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Journal Article↗

PubMed2006

Magnetic nanoparticles: inner ear targeted molecule delivery and middle ear implant.

Superparamagnetic iron oxide nanoparticles (SNP) composed of magnetite (Fe(3)O(4)) were studied preliminarily as vehicles for therapeutic molecule delivery to the inner ear and as a middle ear implant capable of producing biomechanically relevant forces for auditory function. Magnetite SNP were synthesized, then encapsulated in either silica or poly (D,L,-Lactide-co-glycolide) or obtained commercially with coatings of oleic acid or dextran. Permanent magnetic fields generated forces sufficient to pull them across tissue in several round window membrane models (in vitrocell culture, in vivo rat and guinea pig, and human temporal bone) or to embed them in middle ear epithelia. Biocompatibility was investigated by light and electron microscopy, cell culture kinetics, and hair cell survival in organotypic cell culture and no measurable toxicity was found. A sinusoidal magnetic field applied to guinea pigs with SNP implanted in the middle ear resulted in displacements of the middle ear comparable to 90 dB SPL.

Animals↗

PubMed2004

Mitochondrial dysfunction in hearing loss.

Mitochondrial pathology plays an important role in both inherited and acquired hearing loss. Inherited mitochondrial DNA mutations have been implicated in both syndromic and non-syndromic hearing loss, as well as in predisposition to aminoglycoside ototoxicity. Acquired mitochondrial dysfunction in the absence of mitochondrial DNA mutations has also been proposed as playing an important role in noise-induced and toxin-induced hearing loss. Presbycusis, the hearing loss associated with aging, may be caused by mitochondrial dysfunction resulting from the accumulation of acquired mitochondrial DNA mutations and other factors. The pathophysiological mechanisms and clinical implications of these findings are discussed.

Journal Article↗

PubMed2004

Choline acetyltransferase: regulation and coupling with protein kinase and vesicular acetylcholine transporter on synaptic vesicles.

Both the membrane-bound choline acetyltransferase (MChAT) and soluble ChAT (SChAT) were found to be activated by ATP-mediated protein phosphorylation. ATP activation of MChAT but not SChAT was found to depend on the integrity of proton gradient of synaptic vesicles because conditions disrupting the proton gradient also abolished the activation of MChAT by ATP. Among the kinases studied, Ca2+/calmodulin kinase II is most effective in activation of MChAT. Transport of ACh into synaptic vesicles by vesicular acetylcholine transporter (VAChT) is also proton gradient-dependent; therefore we proposed that there is a functional coupling between ACh synthesis and its packaging into synaptic vesicles. This notion is supported by the following findings: first, the newly synthesized [3H]-ACh from [3H]-choline was taken up much more efficiently than the pre-existing ACh; second, ATP-activation of MChAT was abolished when VAChT was inhibited by the specific inhibitor vesamicol; third, the activity of ChAT was found to be markedly increased when neurons are under depolarizing conditions.

Adenosine Triphosphate↗

PubMed2003

Effect of trans-bullar gentamicin treatment on guinea pig angular and linear vestibulo-ocular reflexes.

This study provides the first systematic examination of the effects of intratympanic gentamicin instillation on vestibulo-ocular responses of guinea pigs during both Earth-vertical yaw axis and off-vertical axis rotation. A scleral search coil was sutured to the right eye of pigmented female guinea pigs prior to trans-bullar instillation of a 0.2-ml bolus of either 20 mg/ml or 40 mg/ml of gentamicin (1) into the right middle ear (unilateral treatment groups) or (2) into both ears (bilateral treatment groups). Two weeks later, eye movement responses were tested during yaw axis sinusoidal rotation at 7 frequencies (0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 Hz, 40 deg/s peak velocity) and during off-vertical axis rotation at five constant velocities (20, 40, 60, 80 and 100 deg/s), tilted 30 deg relative to the earth-vertical axis. The main result was that unilateral trans-bullar gentamicin instillation produced almost exclusively unidirectional deficits in horizontal angular vestibulocular reflex (HVOR) responses and modulation and bias responses to off-vertical axis rotation (OVAR). The HVOR gain was reduced during rotation toward the injected ear in a dose-dependent manner for frequencies of 1 Hz and lower, but there was no effect on responses during rotation toward the intact ear. Further, the modulation and bias responses to OVAR were reduced profoundly in a dose-dependent manner during rotation toward the treated ear. It is suggested that these effects indicate selective cytotoxic and/or physiologic effects of gentamicin intoxication in the inner ear or, possibly, the vestibular nerve and central nervous system.

Animals↗

PubMed2003

Demonstration of functional coupling between gamma -aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles.

l-Glutamic acid decarboxylase (GAD) exists as both membrane-associated and soluble forms in the mammalian brain. Here, we propose that there is a functional and structural coupling between the synthesis of gamma-aminobutyric acid (GABA) by membrane-associated GAD and its packaging into synaptic vesicles (SVs) by vesicular GABA transporter (VGAT). This notion is supported by the following observations. First, newly synthesized [(3)H]GABA from [(3)H]l-glutamate by membrane-associated GAD is taken up preferentially over preexisting GABA by using immunoaffinity-purified GABAergic SVs. Second, the activity of SV-associated GAD and VGAT seems to be coupled because inhibition of GAD also decreases VGAT activity. Third, VGAT and SV-associated Ca(2+)calmodulin-dependent kinase II have been found to form a protein complex with GAD. A model is also proposed to link the neuronal stimulation to enhanced synthesis and packaging of GABA into SVs.

Animals↗

PubMed2003

Transtympanic management of tinnitus.

Transtympanic therapy is becoming and important treatment modality for many inner ear disorders. The current therapies aimed at Meniere's disease, sudden sensorineural hearing loss, noise-induced hearing loss, and the tinnitus associated with these disorders and idiopathic tinnitus, however, represents simply an evolutionary step in this treatment modality and must be validated by further scientific study. A number of promising developments including newer more targeted neuroactive medicines, a better understanding of medicine delivery, and the knowledge of the site, origin, and pathophysiology of the symptoms complex will make this therapy more effective. In the future it is possible that many inner ear disorders will be amenable to inner ear medical therapy. Ideally in the future with knowledge of the disease and its etiology the physician will simply pick the established medicine, the established dose, and the established route of administration and achieve a relatively predictable result.

Anti-Bacterial Agents↗

PubMed2003

Vestibular testing abnormalities in individuals with motion sickness.

HYPOTHESIS: The goal of this study was to compare the results of vestibular testing in individuals with motion sickness to a group of control subjects. BACKGROUND: Studying motion sickness is difficult, because no animal model has been developed and symptoms rarely occur outside motion environments. Tests that can be performed in normal laboratory settings, which help to identify individuals with motion sickness, may be valuable in characterizing this disorder. METHODS: Twenty active duty military individuals with well-documented motion sickness were tested. The test battery included sinusoidal rotational chair testing to calculate vestibulo-ocular reflex function, step-velocity testing to calculate vestibular time constants, and posturography testing to assess vestibulo-spinal reflex status. The results of this test battery were compared with a set of age- and sex-matched controls without motion sickness. RESULTS: Vestibular test abnormalities were demonstrated in individuals with motion sickness. Vestibulo-spinal reflex function on posturography was normal in the control group but abnormal in 70% of the individuals with motion sickness. In addition, 5% of the control group demonstrated a minimal shortening of the absolute time constant, whereas 60% of the individuals with motion sickness had abnormal absolute time constants. CONCLUSION: A significant percentage of individuals with motion sickness demonstrate abnormalities in their time constant or vestibulo-spinal reflex function. These abnormalities can be detected using standard, land-based vestibular tests. These preliminary results have implications in understanding the etiology of motion sickness and may provide outcome measures to be used in treating motion sickness.

Adult↗

PubMed2002

Candidate's thesis: enhancing intrinsic cochlear stress defenses to reduce noise-induced hearing loss.

OBJECTIVES/HYPOTHESIS: Oxidative stress plays a substantial role in the genesis of noise-induced cochlear injury that causes permanent hearing loss. We present the results of three different approaches to enhance intrinsic cochlear defense mechanisms against oxidative stress. This article explores, through the following set of hypotheses, some of the postulated causes of noise-induced cochlear oxidative stress (NICOS) and how noise-induced cochlear damage may be reduced pharmacologically. 1) NICOS is in part related to defects in mitochondrial bioenergetics and biogenesis. Therefore, NICOS can be reduced by acetyl-L carnitine (ALCAR), an endogenous mitochondrial membrane compound that helps maintain mitochondrial bioenergetics and biogenesis in the face of oxidative stress. 2) A contributing factor in NICOS injury is glutamate excitotoxicity, which can be reduced by antagonizing the action of cochlear -methyl-D-aspartate (NMDA) receptors using carbamathione, which acts as a glutamate antagonist. 3) Noise-induced hearing loss (NIHL) may be characterized as a cochlear-reduced glutathione (GSH) deficiency state; therefore, strategies to enhance cochlear GSH levels may reduce noise-induced cochlear injury. The objective of this study was to document the reduction in noise-induced hearing and hair cell loss, following application of ALCAR, carbamathione, and a GSH repletion drug D-methionine (MET), to a model of noise-induced hearing loss. STUDY DESIGN: This was a prospective, blinded observer study using the above-listed agents as modulators of the noise-induced cochlear injury response in the species chinchilla langier. METHODS: Adult chinchilla langier had baseline-hearing thresholds determined by auditory brainstem response (ABR) recording. The animals then received injections of saline or saline plus active experimental compound starting before and continuing after a 6-hour 105 dB SPL continuous 4-kHz octave band noise exposure. ABRs were obtained immediately after noise exposure and weekly for 3 weeks. After euthanization, cochlear hair cell counts were obtained and analyzed. RESULTS ALCAR administration reduced noise-induced threshold shifts. Three weeks after noise exposure, no threshold shift at 2 to 4 kHz and <10 dB threshold shifts were seen at 6 to 8 kHz in ALCAR-treated animals compared with 30 to 35 dB in control animals. ALCAR treatment reduced both inner and outer hair cell loss. OHC loss averaged <10% for the 4- to 10-kHz region in ALCAR-treated animals and 60% in saline-injected-noise-exposed control animals. Noise-induced threshold shifts were also reduced in carbamathione-treated animals. At 3 weeks, threshold shifts averaged 15 dB or less at all frequencies in treated animals and 30 to 35 dB in control animals. Averaged OHC losses were 30% to 40% in carbamathione-treated animals and 60% in control animals. IHC losses were 5% in the 4- to 10-kHz region in treated animals and 10% to 20% in control animals. MET administration reduced noise-induced threshold shifts. ANOVA revealed a significant difference (P <.001). Mean OHC and IHC losses were also significantly reduced (P <.001). CONCLUSIONS: These data lend further support to the growing body of evidence that oxidative stress, generated in part by glutamate excitotoxicity, impaired mitochondrial function and GSH depletion causes cochlear injury induced by noise. Enhancing the cellular oxidative stress defense pathways in the cochlea eliminates noise-induced cochlear injury. The data also suggest strategies for therapeutic intervention to reduce NIHL clinically.

Acetylcarnitine↗

PubMed2002

The early kinetics of gentamicin uptake into the inner ear.

Transtympanic gentamicin administration has become a popular modality in the treatment of Ménière's disease. This modality and other inner-ear medical therapy are gaining increased clinical and scientific attention. We previously described the kinetics and effects of gentamicin uptake into the inner ear after delivery of the medicine into the middle ear using a variety of different techniques and sustained-release modalities [1]. In our previous work, we reported an early peak perilymph concentration and the presence of intracellular gentamicin at the 4-hour time point. We also demonstrated the activation of inner-ear damage pathways at this early time point. In this report, we examine the kinetics of gentamicin at very early time points, 1 and 2 hours after administration. Healthy adult chinchillas underwent implantation of middle-ear sustained-release devices (one to each ear) containing gentamicin. The animals then were maintained in a neutral position and underwent perilymph gentamicin sampling at the two predetermined time points. This technique allowed us to assess accurately very early time point inner-ear gentamicin kinetics and to compare the activity. The samples then were run for concentration using mass spectrometry. The information gained from this study may increase our scientific understanding about the effects of gentamicin on the inner ear and may allow clinicians to treat patients more effectively for inner-ear disorders.

Animals↗