Kanamycin Treatment Research Articles

Infection and antibiotic-associated changes in the fecal microbiota of C. rodentium ϕstx2dact-infected C57BL/6 mice.

Enterohemorrhagic Escherichia coli causes watery to bloody diarrhea, which may progress to hemorrhagic colitis and hemolytic-uremic syndrome. While early studies suggested that antibiotic treatment may worsen the pathology of an enterohemorrhagic Escherichia coli (EHEC) infection, recent work has shown that certain non-Shiga toxin-inducing antibiotics avert disease progression. Unfortunately, both intestinal bacterial infections and antibiotic treatment are associated with dysbiosis. This can alleviate colonization resistance, facilitate secondary infections, and potentially lead to more severe illness. To address the consequences in the context of an EHEC infection, we used the established mouse infection model organism Citrobacter rodentium ϕstx2dact and monitored changes in fecal microbiota composition during infection and antibiotic treatment. C. rodentium ϕstx2dact infection resulted in minor changes compared to antibiotic treatment. The infection caused clear alterations in the microbial community, leading mainly to a reduction of Muribaculaceae and a transient increase in Enterobacteriaceae distinct from Citrobacter. Antibiotic treatments of the infection resulted in marked and distinct variations in microbiota composition, diversity, and dispersion. Enrofloxacin and trimethoprim/sulfamethoxazole, which did not prevent Shiga toxin-mediated organ damage, had the least disruptive effects on the intestinal microbiota, while kanamycin and tetracycline, which rapidly cleared the infection without causing organ damage, caused a severe reduction in diversity. Kanamycin treatment resulted in the depletion of all but Bacteroidetes genera, whereas tetracycline effects on Clostridia were less severe. Together, these data highlight the need to address the impact of individual antibiotics in the clinical care of life-threatening infections and consider microbiota-regenerating therapies.IMPORTANCEUnderstanding the impact of antibiotic treatment on EHEC infections is crucial for appropriate clinical care. While discouraged by early studies, recent findings suggest certain antibiotics can impede disease progression. Here, we investigated the impact of individual antibiotics on the fecal microbiota in the context of an established EHEC mouse model using C. rodentium ϕstx2dact. The infection caused significant variations in the microbiota, leading to a transient increase in Enterobacteriaceae distinct from Citrobacter. However, these effects were minor compared to those observed for antibiotic treatments. Indeed, antibiotics that most efficiently cleared the infection also had the most detrimental effect on the fecal microbiota, causing a substantial reduction in microbial diversity. Conversely, antibiotics showing adverse effects or incomplete bacterial clearance had a reduced impact on microbiota composition and diversity. Taken together, our findings emphasize the delicate balance required to weigh the harmful effects of infection and antibiosis in treatment.

Investigation of METTL3 as an inhibitor of kanamycin-induced ototoxicity via stress granule formation.

Methyltransferase-like 3 (METTL3), a component of the N6-methyladenosine (m6A) methyltransferase family, exhibits significant expression in HEI-OC1 cells and cochlear explants. Aminoglycoside antibiotics, known for their ototoxic potential, frequently induce irreversible auditory damage in hair cells, predominantly through oxidative stress mechanisms. However, the specific role of METTL3 in kanamycin-induced hair cell loss remains unclear. This study aims to elucidate the mechanisms by which METTL3 contributes to kanamycin-induced ototoxicity. In vivo experiments demonstrated a notable reduction in METTL3 expression within cochlear explants following kanamycin administration, concomitant with the formation of stress granules (SGs). Similarly, a 24-hour kanamycin treatment led to decreased METTL3 expression and induced SG formation both in HEI-OC1 cells and neonatal cochlear explants, corroborating the in vivo observations. Lentivirus-mediated transfection was employed to overexpress and knockdown METTL3 in HEI-OC1 cells. Knockdown of METTL3 resulted in increased reactive oxygen species (ROS) levels and apoptosis induced by kanamycin, while concurrently reducing SG formation. Conversely, overexpression of METTL3 attenuated ROS generation, decreased apoptosis rates, and promoted SG formation induced by kanamycin. Therefore, METTL3-mediated SG formation presents a promising target for mitigating kanamycin-induced ROS generation and the rate of apoptosis. This finding indicates that METTL3-mediated SG formation holds potential in mitigating kanamycin-induced impairments in cochlear hair cells by reducing ROS formation and apoptosis rates.

Diadenosine tetraphosphate modulated quorum sensing in bacteria treated with kanamycin

BackgroundThe dinucleotide alarmone diadenosine tetraphosphate (Ap4A), which is found in cells, has been shown to affect the survival of bacteria under stress.ResultsHere, we labeled Ap4A with biotin and incubated the labeled Ap4A with the total proteins extracted from kanamycin-treated Escherichia coli to identify the Ap4A binding protein in bacteria treated with kanamycin. Liquid chromatography‒mass spectrometry (LCMS) and bioinformatics were used to identify novel proteins that Ap4A interacts with that are involved in biofilm formation, quorum sensing, and lipopolysaccharide biosynthesis pathways. Then, we used the apaH knockout strain of E. coli K12-MG1655, which had increased intracellular Ap4A, to demonstrate that Ap4A affected the expression of genes in these three pathways. We also found that the swarming motility of the apaH mutant strain was reduced compared with that of the wild-type strain, and under kanamycin treatment, the biofilm formation of the mutant strain decreased.ConclusionsThese results showed that Ap4A can reduce the survival rate of bacteria treated with kanamycin by regulating quorum sensing (QS). These effects can expand the application of kanamycin combinations in the treatment of multidrug-resistant bacteria.

Kanamycin treatment in the pre-symptomatic stage of a Drosophila PD model prevents the onset of non-motor alterations

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor alterations, which is preceded by a prodromal stage where non-motor symptoms are observed. Over recent years, it has become evident that this disorder involves other organs that communicate with the brain like the gut. Importantly, the microbial community that lives in the gut plays a key role in this communication, the so-called microbiota-gut-brain axis. Alterations in this axis have been associated to several disorders including PD.Here we proposed that the gut microbiota is different in the presymptomatic stage of a Drosophila model for PD, the Pink1B9 mutant fly, as compared to that observed in control animals. Our results show this is the case: there is basal dysbiosis in mutant animals evidenced by substantial difference in the composition of midgut microbiota in 8–9 days old Pink1B9 mutant flies as compared with control animals. Further, we fed young adult control and mutant flies kanamycin and analyzed motor and non-motor behavioral parameters in these animals. Data show that kanamycin treatment induces the recovery of some of the non-motor parameters altered in the pre-motor stage of the PD fly model, while there is no substantial change in locomotor parameters recorded at this stage. On the other hand, our results show that feeding young animals the antibiotic, results in a long-lasting improvement of locomotion in control flies.Our data support that manipulations of gut microbiota in young animals could have beneficial effects on PD progression and age-dependent motor impairments.This article is part of the Special Issue on “Microbiome & the Brain: Mechanisms & Maladies”.

The Effect of the Gallbladder Environment during Chronic Infection on Salmonella Persister Cell Formation.

Typhoid fever is caused by Salmonella enterica serovar Typhi (S. Typhi). Around 3-5% of individuals infected become chronic carriers, with the gallbladder (GB) as the predominant site of persistence. Gallstones (GS) aid in the development and maintenance of GB carriage, serving as a substrate to which Salmonellae attach and form a biofilm. This biofilm matrix protects bacteria from the host immune system and environmental stress. This shielded environment is an ideal place for the development of persister cells, a transient phenotype of a subset of cells within a population that allows survival after antibiotic treatment. Persisters can also arise in response to harsh environments such as the GB. Here we investigate if GB conditions affect the number of persisters in a Salmonella population. To simulate the chronic GB environment, we cultured biofilms in cholesterol-coated 96-well plates in the presence of ox or human bile. We then treated planktonic or biofilm Salmonella cultures with high concentrations of different antibiotics. This study suggests that biofilms provide a niche for persister cells, but GB conditions either play no role or have a negative influence on persister formation, especially after kanamycin treatment. The antibiotic target was important, as antimicrobials directed against DNA replication or the cell wall had no effect on persister cell formation. Interestingly, repeated treatment with ciprofloxacin increased the percentage of S. Typhimurium persisters in a biofilm, but this increase was abolished by GB conditions. On the other hand, repeated ciprofloxacin treatment of S. Typhi biofilms in GB conditions slightly increased the fraction of persisters. Thus, while the harsh conditions in the GB would be thought to give rise to increased persisters, therefore contributing to the development of chronic carriage, these data suggest persister cell formation is dampened in this environment.

Quantitative proteomics reveals the antibiotics adaptation mechanism of Aeromonas hydrophila under kanamycin stress

Aeromonas hydrophila is a widespread opportunistic pathogen of aquatic fishes in freshwater habitats. The current emergence of antimicrobial-resistant A. hydrophila has been reported in the world while the bacterial antibiotics adaptive mechanism remains poorly explored. In this study, using quantitative proteomics technology, the behavior of A. hydrophila was investigated by comparing the differentially expression proteins between with and without kanamycin (KAN) treatment. A total of 374 altered proteins including 184 increasing and 190 proteins decreasing abundances were quantified when responding to KAN stress. The bioinformatics analysis showed that stress related proteins were hub proteins that significantly increased to reduce the pressure from the misreading of mRNA caused by KAN. Moreover, several metallic pathways, such as oxidative phosphorylation and TCA cycle pathways may affect KAN resistance. Finally, eight selected genes were deleted and their antibiotics susceptibilities to kanamycin were valued, respectively. Results showed that OmpA II family protein A0KI26, and two-component system protein AtoC may involve in the KAN resistance in this study. In general, our results provide an insight into the behaviors of bacterial responding to KAN stress, and demonstrate the intrinsic antibiotics adaptive mechanism of A. hydrophila. Biological significanceIn this study, the differentially expressed proteins (DEPs) of A. hydrophila strain between with and without kanamycin (KAN) were compared by using a data-independent acquisition (DIA) - based quantitative proteomics method. Bioinformatics analysis showed that stress - related proteins are hub proteins that significantly increased under KAN stress. Moreover, several metallic pathways, such as oxidative phosphorylation and citrate cycle (TCA cycle) pathways, can affect KAN resistance. Finally, our antibiotics susceptibility assay showed that the protein A0KI26 of the OmpA II family, and the AtoC of the two-component system may involve in KAN resistance in this study. These results provide insights into the antibiotics adaptation mechanism of A. hydrophila when responding to KAN stress.

Standardization of efficient regeneration protocol and agrobacterium mediated genetic transformation in RPBio-226 variety of rice (Oryza sativa)

Studies related to the genome engineering in plants require an efficient protocol with high frequency callus induction and regeneration. This present study was conducted at PRR Biotech laboratory, Hyderabad in the year 2020 and aimed to develop an efficient protocol to achieve a high frequency callus induction and genetic transformation of RPBio-226, a Samba Mahsuri rice variety. In-vitro callus induction was carried out using MS media supplemented with different concentrations of 2,4-D viz., 1.0, 1.5, 2.0, 2.5 mg/L singly and in combinations with BAP viz., 0.1, 0.2, 0.4, 0.6 mg/L. Shoot induction was carried out using MS media supplemented with BAP 1.0, 1.5, 2.0, 2.5 mg/L and Kinetin 0.5, 1.0, 1.5, 2.0 mg/L and the Best response observed with 2,4-D and BAP at 2.0 and 0.1 mg/L, respectively. Agrobacterium tumefaciens strain EHA105 harboring PBI 121 plasmid (binary vector) was used in the process of rice transformation. An experiment was planned to use various parameters viz. 0.1, 0.2, 0.3, 0.4 and 0.5 ODs of Agrobacterium cells at 600nm. Embryogenic calli were co-cultivated with Agrobacterium supplemented with different concentrations of acetosyringone (AS) (0, 50, 100 and 150 μM) and 100 μM was found to give the best response for transformation. After Agrobacterium infection, infected calli were selected on the selection media with kanamycin as selection agent. The best hormone for shoot induction was BAP at 1.5 mg/L and kinetin at 0.5 mg/L and highest number of roots was achieved with 0.1 mg/L NAA in combination with 0.2 mg/L IBA. Regenerated plantlets were successfully acclimatized on soilrite mix showing the highest survival rate after 4 weeks of acclimatization. The npt II and Gus specific primers were used for PCR to identify stably transformed progeny that survived kanamycin treatment. GUS Histochemical Analysis of Callus was also carried out for the confirmation of transformation which helps us to generate transgenic plants in future.

Kanamycin Supplement for the Disaggregation of Platelet Clumps in EDTA-Dependent Pseudothrombocytopenia Specimens.

To indicate the ability to disaggregate platelet clumps by vortex mixing and kanamycin supplementation in EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) specimens. For patients with EDTA-PTCP, citrate-anticoagulated, primary EDTA-anticoagulated, vortex-mixed, and kanamycin-treated specimens were tested for complete blood count and platelet-related parameters. Forty-eight specimens were included. Nineteen (39.6%) of the vortex-mixed specimens and 42 (87.5%) of the kanamycin-treated specimens revealed platelet counts more than those of the primary EDTA specimens, with levels exceeding 100 × 109/L. The platelet count and platelet recovery of the kanamycin-treated specimens were higher than those of the vortex-mixed specimens. Kanamycin supplementation to EDTA-PTCP blood may be considered as an alternative approach when the recollection of specimens is impractical. Only platelet-related parameters from kanamycin treatment were suitable for guiding patient management. Further studies about the impact of these methods in patients with various conditions, such as in patients with advanced kidney disease, should be conducted.

Systemic toll-like receptor 9 agonist CpG oligodeoxynucleotides exacerbates aminoglycoside ototoxicity

The Toll-like receptor (TLR) signaling pathway is the key regulator of the innate immune system in response to systemic infection. Several studies have reported that the systemic TLR4 agonist lipopolysaccharide exacerbates aminoglycoside ototoxicity, but the influence of virus-associated TLR7 and TLR9 signaling cascades on the cochlea is unclear. The present study aimed to investigate the auditory effects of systemic TLR7 and TLR9 agonists during chronic kanamycin treatment. CBA/CaJ mice received the TLR7 agonist gardiquimod or TLR9 agonist CpG oligodeoxynucleotides (ODN) one day before kanamycin injection and on the 5th and 10th days during a 14-day course of kanamycin treatment. We observed that systemic gardiquimod or CpG ODN alone did not affect the baseline auditory brainstem response (ABR) threshold. Three weeks after kanamycin treatment, gardiquimod did not significantly change ABR threshold shifts, whereas CpG ODN significantly increased kanamycin-induced ABR threshold shifts. Furthermore, outer hair cell (OHC) evaluation revealed that CpG ODN reduced distortion product otoacoustic emission amplitudes and increased kanamycin-induced OHC loss. CpG ODN significantly elevated cochlear Irf-7, Tnf-α, Il-1, and Il-6 transcript levels. In addition, an increased number of Iba-1+ cells, which represented activated macrophages, was observed in the cochlea treated with CpG ODN. Our results indicated that systemic CpG ODN exacerbated kanamycin-induced ototoxicity and increased cochlear inflammation. This study implies that patients with underlying virus infection may experience more severe aminoglycoside-induced hearing loss if it occurs.

Fursultiamine Prevents Drug-Induced Ototoxicity by Reducing Accumulation of Reactive Oxygen Species in Mouse Cochlea.

Drug-induced hearing loss is a major type of acquired sensorineural hearing loss. Cisplatin and aminoglycoside antibiotics have been known to cause ototoxicity, and excessive accumulation of intracellular reactive oxygen species (ROS) are suggested as the common major pathology of cisplatin- and aminoglycoside antibiotics-induced ototoxicity. Fursultiamine, also called thiamine tetrahydrofurfuryl disulfide, is a thiamine disulfide derivative that may have antioxidant effects. To evaluate whether fursultiamine can prevent cisplatin- and kanamycin-induced ototoxicity, we investigated their preventive potential using mouse cochlear explant culture system. Immunofluorescence staining of mouse cochlear hair cells showed that fursultiamine pretreatment reduced cisplatin- and kanamycin-induced damage to both inner and outer hair cells. Fursultiamine attenuated mitochondrial ROS accumulation as evidenced by MitoSOX Red staining and restored mitochondrial membrane potential in a JC-1 assay. In addition, fursultiamine pretreatment reduced active caspase-3 and TUNEL signals after cisplatin or kanamycin treatment, indicating that fursultiamine decreased apoptotic hair cell death. This study is the first to show a protective effect of fursultiamine against cisplatin- and aminoglycoside antibiotics-induced ototoxicity. Our results suggest that fursultiamine could act as an antioxidant and anti-apoptotic agent against mitochondrial oxidative stress.in cochlear hair cells.

Analysis of Age, Gender and H Analysis of Age, Gender and Hearing Loss in Early Stage Drug-Resistant TB Patients at Abdul Wahab Sjahranie Hospital Samarinda

ABSTRACT
 Hearing loss is one of the side effects of using kanamycin. Hearing loss will reduce the ability to receive information and verbal communication which will interfere with daily activities. This study aims to determine the relationship between age, sex on hearing loss and hearing loss in patients with drug-resistant TB during kanamycin treatment. The design of this study was cross sectional using purposive sampling. The number of samples that met the inclusion and exclusion criteria was 12 patients. The results show that hearing loss is not related to age (p = 0.293), sex (p = 1,000), and hearing loss (p = 0.182). Based on the results of this study it can be concluded that there is no relationship between age, sex on hearing loss and hearing loss during kanamycin administration.
 Keywords: Hearing Loss, Age, Sex

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti.

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

Auditory Evaluation with Pure Tone Audiometry and DPOAE in Kanamycin Treatment of Multidrug-Resistant Tuberculosis

Background: Kanamycin treatment is mainly used in multidrug-resistant tuberculosis (MDR-TB) patients. Hearing impairment associated along with the treatment leads to be permanently. Audiology assessments and monitoring may benefits to detect early on hearing loss. This study aimed to determine the evaluation of hearing alteration after kanamycin injection in the first month treatment. Method: We conducted a prospective study, observational longitudinal analytic with pre and post design in patients treated with kanamycin-based regimens in MDR-TB Outpatients Pulmonology Department Dr. Soetomo general hospital Surabaya. Assessment of hearing result using Pure Tone Audiometry and DPOAE, compared between baseline data and after 4 weeks treatments. Statistical analysis for hearing threshold and cochlear dysfunction based on overall frequency and specific frequency DPOAE examination, using Wilcoxon ranks test and Mc Nemar test. Results: A total of 15 patients (8 males and 7 females) with confirmed diagnosis of MDR-TB were included in this study conducted within 3 months in 2018. There were 15 ears represented with based line and compared to 4 weeks after treatment, showed on Audiometry and DPOAE on overall frequencies, found no significant difference (p > 0.05), but in DPOAE at frequency of 10,000 Hertz (Hz) was found a significant difference (p = 0.002). Conclusion: DPOAE at high frequency (10,000 Hz) may benefits for early detection on hearing impairment. A baseline data with DPOAE is recommended, besides Audiometry for ototoxicity monitoring in Kanamycin treatment in MDRTB patients.

Nisin reduces uterine inflammation in rats by modulating concentrations of pro- and anti-inflammatory cytokines.

The Staphylococcus aureus has been found to be associated with clinical endometritis of cow. The result of oral antibiotic remains poor. Therefore, this study investigates the role of nisin in endometritis. The effect of nisin on the growth and cell wall of Saureus were determined in vitro. Besides the blank control group, animals with established post-partum were inoculated with 0.1mL Saureus intravaginally. Two days post-inoculation, the animals were administered nisin (25mg/kg), kanamycin (30mg/kg), and water (model group) for 7days. On the seventh day, serum and uterine organs were obtained for pro- and anti-inflammatory analysis. The uterine tissue samples were weighed, and histopathological analysis was performed. The results showed that nisin had an inhibitory effect on the growth and cell wall formation of Saureus. Nisin and kanamycin treatment prevented a Saureus-induced decrease in pro-inflammatory cytokines and promoted an increase in the level of serum anti-inflammatory cytokines in the endometrium of these animals. Nisin and kanamycin, significantly decreased (P<0.05) the endometritis-induced increases in uterine weight, restored endometrial architecture and significantly (P<0.05) normalized uterine neutrophils to control levels. Additionally, improved levels of B7-2 , IFN-γ, IL-2, and IL-8 were observed when treated with nisin. Our findings suggest that nisin compared favorably with kanamycin in endometritis prevention, suggesting that nisin can be used in Saureus-induced endometritis by protecting the uterus from Saureus infection.

Endoplasmic reticulum stress is involved in spiral ganglion neuron apoptosis following chronic kanamycin-induced deafness.

Aminoglycoside antibiotics-induced hearing loss is a common sensorineural impairment. Spiral ganglion neurons (SGNs) are first-order neurons of the auditory pathway and are critical for the maintenance of normal hearing. In the present study, we investigated the time-course of morphological changes and the degeneration process of spiral ganglion cells (SGCs) following chronic kanamycin-induced deafness and determined whether the endoplasmic reticulum (ER) stress was involved in the degeneration of SGNs. We detected density changes in SGCs and the expressions of Bip, inositol requirement 1 (IRE1)α, activating transcription factor-6α, p-PERK, p-eIF2α, CHOP, and caspase-12 at each time point after kanamycin treatment. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was also performed. The number of SGC deletions reached ∼50% at the 70th day after kanamycin administration and the ER of most SGCs were dilated. The expression of p-PERK, p-eIF2α, p-IRE1α, Bip, caspase-12, and Chop was significantly unregulated after kanamycin treatment. The number of SGCs that were positive for both TUNEL and caspase-12 increased from day 7 to 28. Taken together, these data demonstrate that ER stress was involved in kanamycin-induced apoptosis of SGNs. Kanamycin-induced SGN apoptosis is mediated, at least in part, by ER stress-induced upregulation of CHOP and caspase-12.

Deciphering of microbial community and antibiotic resistance genes in activated sludge reactors under high selective pressure of different antibiotics

Currently, the effects of high antibiotic concentrations on the performance of microbiota and antibiotic resistance genes (ARGs) in activated sludge (AS) process are not well characterized. Lab-scale batch reactors were performed to evaluate the dynamics of microbial community and ARGs in response to six antibiotics at different concentrations using high-throughput sequencing-based 16S rRNA gene and metagenomic analyses. The presence of antibiotics remarkably decreased the microbial diversity, caused a great change of the microbiota structure, and exerted a selective pressure on the enrichment of potential antibiotic resistant bacteria (ARB), such as Arthrobacter, Thauera, Geothrix, Rudaea, Aridibacter, Conexibacter, Terrimonas, etc. High antibiotic selective pressures increased ARG abundance but simultaneously reduced ARG number. In total, 491 ARG subtypes belonging to 20 ARG types were detected and kanamycin treatment showed the highest ARG abundances. A core set of 54 ARG subtypes that accounted for 66.7%–99.6% of the total ARG abundances were shared by all samples. The increase of the abundances of both corresponding and non-corresponding ARGs under a specific antibiotic treatment revealed the collateral effects of antibiotic selective pressure. Microbial community may play an important role in the composition of ARGs. Network analysis indicated that both internal-type and external-type of ARGs exhibited higher non-random co-occurrence incidences and 18 genera were speculated as the possible hosts for multiple ARGs. This study deciphered the profiles and relationships between microbial community and ARGs in AS process treating wastewater with high antibiotic concentrations and could provide helpful guidance for controlling the development and dissemination of ARB and ARGs.

Secondary Degeneration of Auditory Neurons after Topical Aminoglycoside Administration in a Gerbil Model.

Hair cells in the cochlea can be damaged by various causes. Damaged hair cells can lead to additional destruction of parts of the auditory afferent pathway sequentially, which is called secondary degeneration. Recently, researches regarding cochlear implants have been actively carried out for clinical purposes; secondary degeneration in animals is a much more practical model for identifying the prognosis of cochlear implants. However, an appropriate model for this research is not established yet. Thus, we developed a secondary degeneration model using an ototoxic drug. 35 gerbils were separated into four different groups and kanamycin was applied via various approaches. ABR was measured several times after drug administration. SGCs were also counted to identify any secondary degeneration. The results showed that outer and inner HCs were damaged in all kanamycin-treated groups. Twelve weeks after kanamycin treatment, the round window membrane injection group showed severe subject differences in hair cells and SGC damage, whereas the gelfoam group showed consistent and severe damage in hair cells and SGCs. In this study, we successfully induced secondary degeneration in hair cells in a gerbil model. This model can be used for various purposes in the hearing research area either for treatment or for preservation.

Examination of Calcium-Binding Protein Expression in the Inner Ear of Wild-Type, Heterozygous and Homozygous Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)-Knockout Mice in Kanamycin-Induced Ototoxicity

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with diverse biological effects. It also occurs and exerts protective effects in sensory organs; however, little is known about its effects in the auditory system. Recently, we have shown that PACAP protects cochlear cells against oxidative-stress-induced apoptosis and homozygous PACAP-deficient animals show stronger expression of Ca(2+)-binding proteins in the hair cells of the inner ear, but there are no data about the consequences of the lack of endogenous PACAP in different ototoxic insults such as aminoglycoside-induced toxicity. In this study, we examined the effect of kanamycin treatment on Ca(2+)-binding protein expression in hair cells of wild-type, heterozygous and homozygous PACAP-deficient mice. We treated 5-day-old mice with kanamycin, and 2days later, we examined the Ca(2+)-binding protein expression of the hair cells with immunohistochemistry. We found stronger expression of Ca(2+)-binding proteins in the hair cells of control heterozygous and homozygous PACAP-deficient mice compared with wild-type animals. Kanamycin induced a significant increase in Ca(2+)-binding protein expression in wild-type and heterozygous PACAP-deficient mice, but the baseline higher expression in homozygous PACAP-deficient mice did not show further changes after the treatment. Elevated endolymphatic Ca(2+) is deleterious for the cochlear function, against which the high concentration of Ca(2+)-buffers in hair cells may protect. Meanwhile, the increased immunoreactivity of Ca(2+)-binding proteins in the absence of PACAP provide further evidence for the important protective role of PACAP in ototoxicity, but further investigations are necessary to examine the exact role of endogenous PACAP in ototoxic insults.

Mitochondrial Peroxiredoxin 3 Regulates Sensory Cell Survival in the Cochlea

This study delineates the role of peroxiredoxin 3 (Prx3) in hair cell death induced by several etiologies of acquired hearing loss (noise trauma, aminoglycoside treatment, age). In vivo, Prx3 transiently increased in mouse cochlear hair cells after traumatic noise exposure, kanamycin treatment, or with progressing age before any cell loss occurred; when Prx3 declined, hair cell loss began. Maintenance of high Prx3 levels via treatment with the radical scavenger 2,3-dihydroxybenzoate prevented kanamycin-induced hair cell death. Conversely, reducing Prx3 levels with Prx3 siRNA increased the severity of noise-induced trauma. In mouse organ of Corti explants, reactive oxygen species and levels of Prx3 mRNA and protein increased concomitantly at early times of drug challenge. When Prx3 levels declined after prolonged treatment, hair cells began to die. The radical scavenger p-phenylenediamine maintained Prx3 levels and attenuated gentamicin-induced hair cell death. Our results suggest that Prx3 is up-regulated in response to oxidative stress and that maintenance of Prx3 levels in hair cells is a critical factor in their susceptibility to acquired hearing loss.

Reversible neurotoxicity of kanamycin on dorsal cochlear nucleus

The time course of aminoglycoside neurotoxic effect on cochlear nucleus is still obscure. We examined dynamic pathological changes of dorsal cochlear nucleus (DCN) and investigated whether apoptosis or autophagy was upregulated in the neurotoxic course of kanamycin on DCN after kanamycin treatment. Rats were treated with kanamycin sulfate/kg/day at a dose of 500mg by subcutaneous injection for 10 days. Dynamic pathological changes, neuron density and neuron apoptosis of the DCN were examined at 1, 7, 14, 28, 56, 70 and 140 days after kanamycin treatment. The expressions of JNK1, DAPK2, Bcl-2, p-Bcl-2, Caspase-3, LC3B and Beclin-1 were also detected. Under transmission electron microscopy, the mitochondrial swelling and focal vacuoles as well as endoplasmic reticulum dilation were progressively aggravated from 1 day to 14 days, and gradually recovered from 28 days to 140 days. Meanwhile, both autophagosomes and autolysosomes were increased from 1 day to 56 days. Only few neurons were positive to the TUNEL staining. Moreover, neither the expressions of caspase-3 and DAPK2 nor neurons density of DCN changed significantly. LC3-II was drastically increased at 7 days. Beclin-1 was upgraded at 1 and 7 days. P-Bcl-2 increased at 1, 7, 14 and 28 days. JNK1 increased at 7 days, and Bcl-2 was downgraded at 140 days. LC3-B positive neurons were increased at 1, 7 and 14 days. These data demonstrated that the neurons damage of the DCN caused by kanamycin was reversible and autophagy was upregulated in the neurotoxic course of kanamycin on DCN through JNK1-mediated phosphorylation of Bcl-2 pathway.