[Comparison of the intact and segmented dissection for surface preparation of the cochlea in mice].

Objective: To compare intact dissection and segmented dissection of cochlear surface preparation in adult mice. Methods: From February to March, 2019, Six adult C57BL/6 mice were randomly divided into 2 groups: one group (3 mice) for the intact dissection while the other group (3 mice) for the segmented dissection. Cochlear hair cells were labeled with phalloidin for evaluation of the integrity of the basilar membrane. Results: The basilar membranes can be completely dissected from the cochlea by two approaches. The average dissection time is (16.33±1.86)min with the intact dissection approach while (23.66±3.88) min with the segmented dissection(t=-4.173, P=0.002). Immunofluorescence analysis showed all cochlear hair cells werevisible and intact in two groups. Conclusion: Cochlear basilar membrane can be dissected intact in a short time through both approaches. The approaches selection is dependent on the purpose of experiment and operators' experience.

Receptor-interacting protein kinases modulate noise-induced sensory hair cell death.

Receptor-interacting protein (RIP) kinases promote the induction of necrotic cell death pathways. Here we investigated signaling pathways in outer hair cells (OHCs) of adult male CBA/J mice exposed to noise that causes permanent threshold shifts, with a particular focus on RIP kinase-regulated necroptosis. One hour after noise exposure, nuclei of OHCs in the basal region of the cochlea displayed both apoptotic and necrotic features. RIP1 and RIP3 protein levels increased and caspase-8 was activated. Treatment with pan-caspase inhibitor ZVAD blocked the activation of caspase-8 and reduced the number of apoptotic nuclei, while increasing levels of RIP1, RIP3, and necrotic OHCs. Conversely, treatment with necrosis inhibitor necrostatin-1 (Nec-1) or RIP3 siRNA (siRIP3) diminished noise-induced increases in RIP1 and RIP3, and decreased necrotic OHC nuclei. This treatment also increased the number of apoptotic nuclei without increasing activation of caspase-8. Consistent with the elevation of levels of RIP1 and RIP3, noise-induced active AMPKα levels increased with ZVAD treatment, but decreased with Nec-1 and siRIP3 treatment. Furthermore, treatment with siRIP3 did not alter the activation of caspase-8, but instead increased activation of caspase-9 and promoted endonuclease G translocation into OHC nuclei. Finally, auditory brainstem response functional measurements and morphological assessment of OHCs showed that ZVAD treatment reduces noise-induced deficits. This protective function is potentiated when combined with siRIP3 treatment. In conclusion, noise-induced OHC apoptosis and necrosis are modulated by caspases and RIP kinases, respectively. Inhibition of either pathway shifts the prevalence of OHC death to the alternative pathway.

Effects of neural stem cells on synaptic proteins and memory in a mouse model of Alzheimer's disease.

Transplanting neural stem cells (NSC) to the damaged brain has been regarded as a potential treatment for neurodegenerative diseases such as Alzheimer's disease (AD), a condition characterized by memory loss. We hypothesized that transplantation of NSC into the hippocampal regions of APP + PS1 transgenic (Tg) mice, a well-established model of AD, would enhance the expression of synaptic proteins, which may be helpful for improving cognitive function. Our results showed that NSC transplantation significantly improved spatial learning and memory function in Tg mice. The results obtained by real-time RT-PCR, immunofluorescence, and Western blot analyses demonstrated that the expression of synaptophysin (SYN) and that of growth-associated protein-43 (GAP-43) in Tg-NSC mice, 8 weeks after transplantation, were significantly improved compared with what was observed in Tg-Veh (control) mice. This finding was confirmed by the increase in the number of synapses in Tg-NSC mice as observed via electron microscopy. Our results suggest that NSC-induced changes can recover memory loss in APP + PS1 transgenic mice, possibly by establishing new neural circuits resulting from the engrafted NSC.

Noise-induced time-dependent changes in oxidative stress in the mouse cochlea and attenuation by D-methionine.

Oxidative stress in the cochlea is considered to play an important role in noise-induced hearing loss. This study determined changes in superoxide dismutase (SOD), catalase, lipid peroxidation (LPO) and the auditory brainstem response (ABR) in the cochlea of C57BL/6 mice prior to and immediately, 1, 3, 7, 10, 14 and 21 days after noise exposure (4 kHz octave band at the intensity of 110 dB SPL for 4 h). A significant increase in SOD activity immediately and on 1st day after noise exposure, without a concomitant increase in catalase activity suggested a difference in the time dependent changes in the scavenging enzymes, which facilitates the increase in LPO observed on day 7. The ABR indicated significant noise-induced functional deficits which stabilized in 2 weeks with a permanent threshold shift (PTS) of 15 dB at both 4 kHz and 8 kHz. The antioxidant D-methionine (D-Met) reversed the noise-induced changes in LPO levels and enzyme activities. It also significantly reduced the PTS observed on the 14th day from 15 dB to 5 dB for 4 kHz. In summary, the findings indicate that time-dependent alterations in scavenging enzymes facilitate the production of reactive oxygen species and that D-met effectively attenuates noise-induced oxidative stress and the associated functional loss in the mouse cochlea.

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.

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.

Overexpression of copper/zinc-superoxide dismutase protects from kanamycin-induced hearing loss.

The participation of reactive oxygen species in aminoglycoside-induced ototoxicity has been deduced from observations that aminoglycoside-iron complexes catalyze the formation of superoxide radicals in vitro and that antioxidants attenuate ototoxicity in vivo. We therefore hypothesized that overexpression of Cu/Zn-superoxide dismutase (h-SOD1) should protect transgenic mice from ototoxicity. Immunocytochemistry confirmed expression of h-SOD1 in inner ear tissues of transgenic C57BL/6-TgN[SOD1]3Cje mice. Transgenic and nontransgenic littermates received kanamycin (400 mg/kg body weight/day) for 10 days beginning on day 10 after birth. Auditory thresholds were tested by evoked auditory brain stem responses at 1 month after birth. In nontransgenic animals, the threshold in the kanamycin-treated group was 45-50 dB higher than in saline-injected controls. In the transgenic group, kanamycin increased the threshold by only 15 dB over the respective controls. The effects were similar at 12 and 24 kHz. The protection by overexpression of superoxide dismutase supports the hypothesis that oxidant stress plays a significant role in aminoglycoside-induced ototoxicity. The results also suggest transgenic animals as suitable models to investigate the underlying mechanisms and possible strategies for prevention.

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.

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.

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.

Salicylate attenuates gentamicin-induced ototoxicity.

Aminoglycosides, primarily gentamicin, are the most commonly used antibiotics worldwide despite their toxicity to the kidney and the inner ear. A preventive therapy against these side effects should combine safety and efficacy with low cost because aminoglycoside-induced deafness is most prevalent in developing countries. We have previously shown that aminoglycosides catalyze the formation of free radicals in an iron-dependent reaction and have delineated the structure of an iron-gentamicin complex. Here we demonstrate that 2-hydroxybenzoate (salicylate), which can act as an iron chelator and antioxidant, effectively protects against gentamicin-induced hearing loss in guinea pigs. Co-therapy with salicylate reduced a profound gentamicin-induced auditory threshold shift of more than 60 dB to less than 20 dB. Morphological assessment of the inner ear confirmed protection of auditory sensory cells. Salicylate altered neither serum levels of gentamicin nor its antibacterial efficacy. Because the required salicylate levels correspond to anti-inflammatory levels in humans, this treatment holds promise for clinical application.

Stimulation of free radical formation by aminoglycoside antibiotics.

We have previously shown gentamicin to form a redox-active iron chelate. This study investigates whether other aminoglycosides can likewise stimulate the generation of reactive oxygen species (free radicals). Kanamycin, neomycin and streptomycin were compared to gentamicin in intact cells and in cell-free in vitro assays using luminescence detection with lucigenin or luminol. Neutrophils and Epstein-Barr virus-transformed lymphoblastoid cells served as cell models in which a respiratory burst of superoxide was induced by phorbol ester. The addition of millimolar amounts of any of the aminoglycosides increased the luminescence significantly. The drugs also increased the formation of free radicals in an enzymatic (hypoxanthine-xanthine oxidase) and a non-enzymatic (phenazine methosulfate-NADH) superoxide-generating system. Half-maximal stimulation was reached with (0.4 mM gentamicin, and there was an absolute requirement for an electron donor, arachidonic acid. In both intact cells and cell-free systems, gentamicin-enhanced luminosity was suppressed by iron chelators. These results demonstrate that different aminoglycoside antibiotics can stimulate the formation of free radicals in biological and in cell-free systems. Luminescence detection is a convenient assay method to investigate the redox properties of these drugs.

Formation of reactive oxygen species following bioactivation of gentamicin.

The present study investigated the ability of gentamicin to catalyze free radical reactions and probed the underlying mechanisms by hydroethidine imaging, oxygen consumption, and reduction of cytochrome c. In Epstein-Barr virus-transformed lymphoblastoid cells, a respiratory burst was induced by phorbol ester and detected by hydroethidine, a fluorescent indicator of superoxide radical. The addition of gentamicin increased the fluorescence two-fold while gentamicin did not produce fluorescence in the absence of phorbol ester. In membrane preparations, gentamicin did not enhance NADPH consumption ruling out a direct activation of NADPH oxidase. The formation of reactive oxygen species by gentamicin was additionally supported by experiments that showed gentamicin increased oxygen consumption two-fold in intact cells and a cell-free system. In addition, generation of superoxide was indicated by the gentamicin-stimulated reduction of cytochrome c. The stimulation by gentamicin depended upon the presence of iron (FeII/FeIII) and of arachidonic acid as an electron donor. These results support the hypothesis that an iron-gentamicin complex can increase reactive oxygen species in nonenzymatic and in biological systems. The requirement for a reductive activation in intact cells (e.g., by a respiratory burst) is interpreted as the conversion of an inactive FeIII-gentamicin to a redox-active FeII-gentamicin complex.

Iron chelators protect from aminoglycoside-induced cochleo- and vestibulo-toxicity.

The attenuation of gentamicin-induced hearing loss by iron chelators and radical scavengers has recently been demonstrated in guinea pig in vivo. The present study investigated whether this protective treatment is effective against hearing loss and vestibular damage caused by other aminoglycosides. In a direct comparison, dihydroxybenzoate was chosen over deferoxamine because of its more effective action against gentamicin-induced hearing loss. Guinea pigs received daily injections of kanamycin (250 mg/kg/d) or streptomycin (300 mg/kg/d) for 23 d to induce severe cochlear or vestibular toxicity, respectively. Kanamycin injections resulted in a progressive threshold shift of 60 to 80 dB at 18 kHz, while streptomycin injections induced only a small threshold shift. In contrast, streptomycin abolished almost all vestibular responses. Coinjection of aminoglycosides with a mixture of dihydroxybenzoate (100 mg/kg/d) and mannitol (30 mg/kg/d) significantly attenuated kanamycin-induced hearing loss and protected against streptomycin-induced vestibulotoxicity. DHB/mannitol did not affect serum levels or the antibacterial efficacy of either aminoglycoside. This study supports the idea that iron and free radicals play a critical role in the toxic side effects of aminoglycoside antibiotics. Furthermore, the previously proposed therapeutic protection is not limited to gentamicin but applicable to other aminoglycosides as well.

Are aminoglycoside antibiotics excitotoxic?

Guinea pigs received gentamicin to induce a profound hearing loss (61 dB auditory threshold shift at 18 kHz). Concomitant administration of maleic or tartaric acid dissolved in dimethyl sulfoxide (DMSO) significantly reduced the threshold shift to < 40 dB. The results have several important implications. First, they support the hypothesis of a free-radical mechanism of gentamicin toxicity since the protective compounds are metal chelators and scavengers. Second, they caution against these and similar chemicals, commonly found in drug preparations, as vehicles in tests of aminoglycoside toxicity. For example, a recent study by others describing attenuation of aminoglycoside ototoxicity by NMDA antagonists may have been influenced by the presence of maleate, tartrate and DMSO. Third, they suggest simple antioxidants as a potentially efficient and inexpensive clinical prophylaxis of aminoglycoside-induced hearing loss.

Prevention of aminoglycoside-induced hearing loss.

This review discusses the current problem of ototoxicity associated with the worldwide use of aminoglycoside antibiotics. Pathology and pathophysiology of cochlear and vestibular damage have long been recognized as a preferential destruction of hair cells beginning in the cochlea with the outer hair cells of the lower turns. This is accompanied by high frequency hearing loss progressing to lower frequencies during and even after treatment with these drugs. A novel hypothesis of the underlying biochemical mechanism is based on the formation of free radicals by an aminoglycoside-iron complex. A protective treatment against aminoglycoside-induced hearing loss by co-administration of iron chelators has been successfully documented in guinea pig. This pharmacological intervention does not change serum levels of aminoglycosides nor their antibacterial efficacy. Since iron chelators are established therapeutic drugs, the proposed treatment should lend itself to clinical application. Aminoglycosides, long established for their high efficacy and low cost, may thus continue to play an important role in combating infectious diseases.