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Biomedical subjects

J Schacht

Publications and source records attributed to J Schacht.

At least 19 recordsLinked to original sources
PubMed2006

Caspase-independent pathways of hair cell death induced by kanamycin in vivo.

Cochlear and vestibular sensory cells undergo apoptosis when exposed to aminoglycoside antibiotics in organ culture, but mechanisms of chronic drug-induced hair cell loss in vivo are unclear. We investigated cell death pathways in a mouse model of progressive kanamycin-induced hair cell loss. Hair cell nuclei showed both apoptotic- and necrotic-like appearances but markers for classic apoptotic pathways (cytochrome c, caspase-9, caspase-3, JNK, TUNEL) were absent. In contrast, drug treatment caused EndoG translocation, activation of mu-calpain, and both the synthesis and activation of cathepsin D. Poly (ADP-ribose) polymerase 1 (PARP1) was decreased, but a caspase-derived 89 kDa PARP1 fragment was not present. The mRNA level of PARP1 remained unchanged. Thus, chronic administration of aminoglycosides causes multiple forms of cell death, without a major contribution by classic apoptosis. These results provide a better understanding of the toxic effects of aminoglycosides and are relevant to design protection from aminoglycoside-induced hearing loss.

Animals↗
PubMed2005

Post-exposure treatment attenuates noise-induced hearing loss.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in sensory cell and neural death in the peripheral nervous system, including damage induced by noise trauma. Antioxidant administration prior to or concomitant with noise exposure can prevent auditory deficits, but the efficacy of a delayed treatment is not known. We have recently found continued reactive oxygen species/reactive nitrogen species formation in the ear for 7-10 days following noise exposure and reasoned that antioxidant intervention during this period should also reduce noise-induced hearing loss. Guinea-pigs were subjected to 4 kHz octave band noise at 120 decibels sound- pressure-level (dB SPL) for 5 hours and received treatment with ROS and RNS scavengers (salicylate and trolox) beginning 3 days prior, 1 hour, 1, 3, or 5 days after noise exposure. Auditory thresholds were assessed by sound-evoked auditory brainstem response at 4, 8, and 16 kHz, before and 10 days after noise exposure. Hair cell damage was analyzed by quantitative histology, and free radical activity was determined immunohistochemically via 4-hydroxynonenal and nitrotyrosine as markers of reactive oxygen species and reactive nitrogen species action. Delivered up to 3 days after noise exposure, salicylate and trolox significantly reduced auditory brainstem response deficits, reduced hair cell damage, and decreased reactive oxygen species and reactive nitrogen species formation. Earlier drug treatment was more effective than later treatment. Our results detail a window of opportunity for rescue from noise trauma, and provide evidence for both morphological and functional protection by delayed pharmacological intervention.

Aldehydes↗
PubMed2004

Modulation of activator protein 1/DNA binding activity by acoustic overstimulation in the guinea-pig cochlea.

Changes in gene expression are part of the homeostatic machinery with which cells respond to external stimuli or assaults. The activity of the early response transcriptional factor activator protein-1 (AP-1) can be modulated by a variety of environmental stimuli including those that alter the cellular oxidation/reduction status. This study investigates the activation of AP-1/DNA binding in the guinea-pig cochlea in response to acoustic overstimulation which produces reactive oxygen species. Electrophoretic mobility shift assays revealed that binding of AP-1 to its radiolabeled oligonucleotide probe markedly changed in nuclear extracts of inner ear tissues following intense noise exposure (4 kHz octave band, 115 dB, 5 h). AP-1/DNA binding increased in the organ of Corti and the lateral wall tissues immediately after the exposure, returning to near-baseline levels 5 h later. At 15 h after noise, a second peak of binding activity occurred in the organ of Corti whereas stria vascularis showed a lesser but more sustained activity. Binding in nuclear extracts from the spiral ganglion did not change. Incubation of nuclear extracts with antibodies against Fos/Jun family proteins prior to a supershift assay showed Fra-2 as a major component of the AP-1 complex immediately after the noise exposure. In the organ of Corti, Fra-2 immunoreactivity was localized to the middle turn, i.e. the region which is most affected by the 4-kHz octave band exposure. The results suggest the modulation of gene expression via the activation of AP-1 as a consequence of noise trauma but also demonstrate differential responses in cochlear tissues.

Acoustic Stimulation↗
PubMed2004

[Aminoglycoside- and cisplatin-ototoxicity: from basic science to clinics].

Gentamicin and cisplatin are clinically widely used pharmacological agents which may induce irreversible hearing loss as a side effect. Concerning the pathomechanisms of ototoxicity as well as preventive strategies there are similarities but also some differences. In this review we focus on the role of reactive oxygen species, the antioxidant system, cellular iron and calcium as well as nitric oxide and neurotrophins on gentamicin- and cisplatin-ototoxicity. Furthermore we deal with apoptotic and necrotic cell death as well as the role of mitochondria in these cell injury processes.

Aminoglycosides↗
PubMed2001

Acetylcholine-evoked calcium increases in Deiters' cells of the guinea pig cochlea suggest alpha9-like receptors.

The medial efferent system innervates outer hair cells in the organ of Corti. Neurotransmission at this synapse is mediated by acetylcholine (ACh) acting on nicotinic ACh receptors containing the alpha9 subunit. In addition to the sensory cells, the supporting cells of the mammalian cochlea also receive efferent innervation but the neurotransmitter(s) at these synapses are not known. We show slow transient increases of intracellular calcium evoked by ACh in isolated Deiters' cells of the guinea pig cochlea. The antagonists atropine, d-tubocurarine and strychnine blocked the ACh-effect. Nicotine was an ineffective agonist. The pharmacologic profile and the kinetics of the calcium response suggest an alpha9-like ACh receptor on Deiters' cells similar but not identical to that on the outer hair cells.

Acetylcholine↗
PubMed2001

ATP and nitric oxide modulate intracellular calcium in isolated pillar cells of the guinea pig cochlea.

Supporting cells in the mammalian cochlea have recently received attention as potential targets of neurotransmitters, neuromodulators, and neurohumoral agents. Calcium homeostasis in Deiters' and Hensen's cells, for example, is regulated by ATP and nitric oxide. We studied the intracellular calcium concentration [Ca2+]i in isolated pillar cells of the guinea pig cochlea in response to extracellular ATP and nitric oxide using the fluorescent indicator fluo-3. [Ca2+]i increased rapidly and significantly throughout the pillar cell in response to a bolus of ATP or 2-methylthio ATP while alpha,beta-methylene ATP was ineffective. The response to ATP was inhibited by suramin and Cibacron Blue but not by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid. This pharmacological profile is consistent with a [Ca2+]i increase largely mediated by P2Y receptors. In Ca2+-free medium supplemented with EGTA, the response to extracellularATP was reduced by 33%, suggesting a contribution of calcium influx to the overall effect. The ATP-induced increase of [Ca2+] was attenuated by NO donors (sodium nitroprusside or diethylamine NONOate), and this attenuation was reversed by KT5823, an antagonist to protein kinase G. The results indicate the involvement of purinergic mechanisms and the nitric oxide/cyclic GMP/protein kinase G pathway in the regulation of [Ca2+]i in cochlear pillar cells.

Adenosine Triphosphate↗
PubMed2001

Differential vulnerability of basal and apical hair cells is based on intrinsic susceptibility to free radicals.

The base of the cochlea is more vulnerable to trauma than the apex as seen in the pattern of hair cell damage by cisplatin or aminoglycosides. The differential vulnerability is maintained in organotypic cultures exposed directly to these drugs, suggesting there may be an intrinsic difference in sensitivity to damage along the cochlear spiral. We therefore investigated the survival capacity of isolated outer hair cells and strips dissected from different turns of the guinea pig organ of Corti in short-term culture. Cells were stained with fluorescent indicators of viable or dead cells, calcein-AM and ethidium homodimer. After 5 h at room temperature, up to 90% of outer hair cells from the apex survived, but less than 30% from the base. In contrast, basal inner hair cells remained viable, and supporting cells survived for at least 20 h. The difference in survival capacity between basal and apical outer hair cells coincided with a significantly lower level of the antioxidant glutathione in basal outer hair cells compared with apical outer hair cells. This suggested that basal outer hair cells may be more vulnerable to free-radical damage than apical outer hair cells. The survival of basal outer hair cells was significantly improved by addition of the radical scavengers n-acetyl cysteine, p-phenylenediamine, glutathione, mannitol or salicylate. The protection by antioxidants implies that the accelerated death of basal outer hair cells is due to free-radical damage. The results support an intrinsic susceptibility to free radicals that differs among cochlear cell populations. This differential provides a rational explanation for base-to-apex gradients observed in various forms of cochlear pathology.

Animals↗
PubMed2001

Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague-Dawley rat.

The availability of genetic information, transgenic and knock-out animals make the mouse a primary model in biomedical research. Aminoglycoside ototoxicity, however, has rarely been studied in mature mice because they are considered highly resistant to the drugs. This study presents models for kanamycin ototoxicity in adult CBA/J, C57BL/6 and BALB/c mouse strains and a comparison to Sprague-Dawley rats. Five-week-old mice were injected subcutaneously twice daily with 400-900 mg kanamycin base/kg body weight for 15 days. Kanamycin induced dose-dependent auditory threshold shifts of up to 70 dB at 24 kHz as measured by auditory brain stem-evoked responses. Vestibular function was also affected in all strains. The functional deficits were accompanied by hair cell loss in both cochlear and vestibular neurosensory epithelia. Concomitant administration of the antioxidant 2,3-dihydroxybenzoate significantly attenuated the kanamycin-induced threshold shifts. In adult male Sprague-Dawley rats, doses of 1 x 500 mg or 2 x 300 mg kanamycin base/kg body weight/day x 14 days induced threshold shifts of approximately 50 dB at 20 kHz. These were accompanied by loss of outer hair cells. The order of susceptibility, BALB>CBA>C57, was not due to differences in the pharmacokinetics of kanamycin. It also did not correlate with the presence of Ahl/Ahl2 genes which predispose C57 and BALB strains, respectively, to accelerated age-related hearing loss. Pigmentation, however, paralleled this rank order suggesting an influence of melanin on cochlear antioxidant status.

Aminoglycosides↗
PubMed2000

Intense noise induces formation of vasoactive lipid peroxidation products in the cochlea.

This study investigates the correlation between the formation of reactive oxygen species (ROS) and auditory damage in noise-induced hearing loss. The noise exposure (4-kHz octave band, 115 dB SPL, 5 h) created permanent threshold shifts at frequencies from 2 to 20 kHz. The lipid peroxidation product, 8-isoprostane, was determined biochemically and histochemically as an indicator of ROS. Noise exposure increased 8-isoprostane levels in the cochlea in a time-dependent manner. After 5 h of exposure, 8-isoprostane levels were more than 30-fold greater than baseline, and decreased rapidly after the termination of noise. The immunoreactivity to 8-isoprostane was increased in the stria vascularis, spiral ganglion cells and the organ of Corti. In the organ of Corti, immunostaining was restricted to the second turn in a region 10-12 mm from the apex. This region sustained most of the permanent hair cell damage as revealed in surface preparations. Outer hair cells were more heavily immunostained than inner hair cells while Hensen's cells showed still less immunostain. These data are consistent with the view that ROS are involved in noise-induced damage. However, the relationship between ROS formation and tissue damage appears complex. In the organ of Corti, the pattern of noise-induced lipid peroxidation correlates well with subsequent morphological damage. The stria vascularis, however, does not sustain permanent damage despite intense lipid peroxidation. Differences in endogenous antioxidant levels and commitment to different apoptotic or survival pathways may underlie such differential responses.

Animals↗
PubMed2000

Nitric oxide/cyclic GMP pathway attenuates ATP-evoked intracellular calcium increase in supporting cells of the guinea pig cochlea.

We demonstrate here that nitric oxide (NO) attenuates ATP-evoked calcium transients in Deiters' and Hensen's cells, "supporting" (nonsensory) cells of the guinea pig cochlea, by means of activation of soluble guanylyl cyclase and protein kinase G. The enzymatic activities associated with the nitric oxide/cGMP/protein kinase G pathway had previously been demonstrated to be present in Deiters' and Hensen's cells. We now isolate these cells and measure changes in intracellular free calcium by using the calcium indicator fluo-3. In Deiters' cells, calcium increased rapidly in response to the application of ATP. The increase was attenuated when the pathway was stimulated by NO donors (diethylamine NONOate or sodium nitroprusside) or the cyclic GMP analog, 8-bromo-cyclic GMP. When the activation of the pathway was blocked by the additional presence of inhibitors of soluble guanylyl cyclase (LY83583) or protein kinase G (Rp-8-bromo-cyclic GMP or KT5823), the response to ATP was restored. The reactions also occurred in calcium-free media. Hensen's cells responded similarly. These results provide evidence that intracellular calcium is regulated by the NO/cGMP/protein kinase G pathway in the inner ear.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Acoustic trauma enhances DNA binding of transcription factor AP-1 in the guinea pig inner ear.

Radiolabeled probes for the transcription factor, activator protein-1 (AP-1), bound differentially to nuclear fractions of discrete structures of the guinea pig cochlea (organ of Corti, lateral wall tissues, and spiral ganglion). Noise exposure (4 kHz octave band, 115 dB, for 5 h) significantly increased AP-1 binding in both the organ of Corti and lateral wall tissues but not in the spiral ganglion. Supershift analysis using an antibody against c-Fos protein demonstrated that enhancement of AP-1/DNA binding was at least in part due to the expression of c-Fos protein. These results suggest that AP-1 is involved in the molecular mechanism(s) mediating noise-induced cochlear damage.

Animals↗
PubMed2000

Antioxidants attenuate gentamicin-induced free radical formation in vitro and ototoxicity in vivo: D-methionine is a potential protectant.

We have recently suggested antioxidant therapy against aminoglycoside-induced hearing loss based on the hypothesis of a redox-active aminoglycoside-iron complex causing ototoxicity. The present study compares seven antioxidants and iron chelators for their ability to attenuate gentamicin-induced free radical generation in vitro and ototoxicity in guinea pig in vivo. Free radical formation by gentamicin was measured by chemiluminescence detection both in a non-enzymatic system in vitro and in cell culture. Deferoxamine, 2,3-dihydroxybenzoate, or salicylic acid suppressed gentamicin-induced luminescence in both tests. This indicated the usefulness of the assay as a screen for potential protectants since these agents had previously been shown to attenuate gentamicin-induced ototoxicity in vivo. Histidine and D-methionine, amino acids with chelating and antioxidant properties, also suppressed gentamicin-mediated luminosity both in vitro and in cell culture. In contrast, the metal chelators succimer (2, 3-dimercaptosuccinic acid (DMSA)) and trientine (N, N'-bis[2-aminoethyl]-1,2 ethanediamine) promoted free radical formation and were excluded from further studies. Histidine and D-methionine were then administered to guinea pigs receiving concurrent treatment with gentamicin (120 mg/kgx19 days). Threshold shifts induced by gentamicin were significantly attenuated by twice-daily injections of D-methionine. Once-daily injections of histidine or D-methionine were less effective, pointing to the importance of pharmacokinetics in antioxidant protection in vivo. The study presents a simple screening system for agents with the potential to attenuate gentamicin-induced hearing loss. It also supports the hypothesis of free radical formation as an underlying cause of gentamicin ototoxicity.

Animals↗
PubMed2000

Glutathione limits noise-induced hearing loss.

The generation of reactive oxygen species (ROS) is thought to be part of the mechanism underlying noise-induced hearing loss (NIHL). Glutathione (GSH) is an important cellular antioxidant that limits cell damage by ROS. In this study, we investigated the effectiveness of a GSH supplement to protect GSH-deficient animals from NIHL. Pigmented guinea pigs were exposed to a 4 kHz octave band noise, 115 dB SPL, for 5 h. Group 1 had a normal diet, while groups 2, 3 and 4 were fed a 7% low protein diet (leading to lowered tissue levels of GSH) for 10 days prior to noise exposure. One hour before, immediately after and 5 h after noise exposure, subjects received either an intraperitoneal injection of 5 ml/kg body weight of 0.9% NaCl (groups 1 and 2), 0.4 M glutathione monoethyl ester (GSHE; group 3) or 0.8 M GSHE (group 4). Auditory thresholds were measured by evoked brain stem response at 2, 4, 8, 12, 16 and 20 kHz before and after noise exposure. Ten days post exposure, group 1 showed noise-induced threshold shifts of approximately 20 dB at 2, 16 and 20 kHz and 35 to 40 dB at other frequencies. Threshold shifts in group 2 were significantly greater than baseline at 2, 4, 16 and 20 kHz. GSHE supplementation in a dose-dependent fashion attenuated the threshold shifts in the low protein diet animals. Hair cell loss, as evaluated with cytocochleograms, was consistent with the auditory-evoked brainstem response results. Group 2 exhibited significantly more hair cell loss than any of the other groups; hair cell loss in group 3 was similar to that seen in group 1; group 4 showed less loss than group 1. These results indicate that GSH is a significant factor in limiting noise-induced cochlear damage. This is compatible with the notion that ROS generation plays a role in NIHL and that antioxidant treatment may be an effective prophylactic intervention.

Animals↗
PubMed2000

Protection from ototoxicity of intraperitoneal gentamicin in guinea pig.

BACKGROUND: Aminoglycoside antibiotics are common to treat peritonitis and exit-site infections in patients on peritoneal dialysis. Ototoxicity (loss of hearing or balance) is a well-documented adverse effect of aminoglycosides, and severe ototoxic reactions have been noted in patients receiving these drugs by intraperitoneal lavage. We have proposed a free-radical hypothesis for the mechanism of aminoglycoside ototoxicity and suggested a therapeutic prevention by the concomitant administration of antioxidants or iron chelators. Here we investigate whether 2, 3-dihydroxybenzoate can prevent the ototoxicity of intraperitoneal gentamicin. METHODS: Two strains of pigmented guinea pigs received daily intraperitoneal injections of gentamicin. Both strains developed ototoxicity, although different dosages were needed to produce similar auditory deficits (120 mg gentamicin base/kg body weight daily for 19 days vs. 135 mg/kg for 14 days). Dihydroxybenzoate was administered intraperitoneally once or twice daily. Auditory thresholds were measured by evoked brain stem response. Pathology was assessed as a loss of sensory cells in surface preparations of the organ of Corti. RESULTS: The auditory threshold shifts and hair cell loss were similar to the pathology observed following subcutaneous injections of gentamicin. Animals sustained almost complete loss of outer hair cells in the basal cochlea and a progressive hearing loss with threshold shifts of 60 dB at 18 kHz. The concomitant administration of dihydroxybenzoate significantly attenuated the threshold shift to less than 30 dB and reduced the loss of hair cells. The treatment with dihydroxybenzoate did not affect serum gentamicin levels. CONCLUSIONS: Antioxidant therapy is a promising approach to prevent aminoglycoside-induced hearing loss following intraperitoneal application.

Animals↗
PubMed2000

Intermolecular and intramolecular reorientations in nonchiral smectic liquid-crystalline phases studied by broadband dielectric spectroscopy

Molecular dynamics has been studied by broadband dielectric relaxation spectroscopy in the Sm-A, Sm-B, and Sm-E phases (Sm denotes smectic) of a homologous series of nonchiral stilbenes. An assignment of modes is presented based on their dependence on temperature and molecular length, and, as far as they obey the Arrhenius law, their activation energy has been determined. In general, reorientations of entire molecules around their short axis are active, whereas reorientations of entire molecules around their long axis are locked out in the Sm-E phase of shorter homologs, yet intramolecular reorientations of polar sites have been established. Strong evidence is presented for an interdependence of reorientations of entire molecules around the short and long axes within the biaxial Sm-E phase of longer homologs.

Journal Article↗
PubMed1999

Antioxidant therapy attenuates aminoglycoside-induced hearing loss.

Aminoglycosides have been a mainstay in antibacterial therapy for over 50 years. Worldwide today, they are the most commonly used antibiotics because of high efficacy and low cost. However, nephrotoxicity and ototoxicity are two major side effects. Prevention of these side effects has been attempted for several decades, but no clinical treatment has yet been realized. A therapeutic approach based on a novel hypothesis of the toxic mechanism of aminoglycosides is proposed. Aminoglycosides chelate iron, and the resulting iron-aminoglycoside complex is redox-active, catalyzing the formation of free radicals. The prevention of ototoxicity with antioxidants and iron chelators has been successfully demonstrated in guinea pigs. Both hearing loss and vestibular dysfunction induced by several aminoglycosides were significantly attenuated without compromising the antibacterial efficacy of the drugs. This finding may lead to a safe and effective way to eliminate the threat of ototoxicity of this commonly used class of antibiotics.

Aminoglycosides↗