Gentamicin-treated Animals Research Articles

Combining nonsense mutation suppression therapy with nonsense-mediated decay inhibition in neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) results from germline mutations in the tumor-suppressor gene NF1 and predisposes patients to developing nervous system tumors. Twenty percent of NF1 patients harbor nonsense mutations resulting in premature termination codons (PTCs). Nonsense suppression therapies can facilitate ribosomal readthrough of PTCs to restore full-length protein, but their potential in NF1 is underexplored. We developed a minipig model of NF1 carrying a PTC to test whether nonsense suppression could restore expression of the NF1-encoded protein neurofibromin invitro and invivo. Nonsense suppression did not reliably increaseneurofibromin in primary NF1-/- Schwann cells isolated from minipig neurofibromas but could reduce phosphorylated ERK. Gentamicin invivo produced a similar plasma pharmacokinetic profile to humans and was detectable in clinicallyrelevant tissues, including cerebral cortex, sciatic nerve,optic nerve, and skin. In gentamicin-treated animals, increased neurofibromin expression was seen in the optic nerve. Nonsense-mediated decay (NMD) causes degradation of transcripts with PTCs, which could impede nonsense suppressiontherapies. Nonsense suppression in combination with NMD inhibition restored neurofibromin protein expression in primary NF1-/- Schwann cells isolated from minipig neurofibromas. Thus, the effectiveness of nonsense suppression therapies can be improved in NF1 by the concurrent use of NMD inhibitors.

Nephroprotective Activity of Ethanolic Extract of Carissa carandas Leaves against Gentamicin-Induced Acute Kidney Injury in Wistar Albino Rats

The present study was aimed to evaluate the nephroprotective activity of ethanolic extract of Carissa carandas Linn. Leaves (EECC) in Gentamicin-induced nephrotoxicity in Wistar albino rats. The renal damage was induced by Gentamicin (80mg/kg body weight, i.p.). Nephroprotective activity was investigated by the administration of EECC at two different doses (100 and 200mg/kg body weight, p.o) for 28 days and by assessing serum parameters, renal oxidative stress markers and histopathological studies. Gentamicin-treated animals showed an increase in serum creatinine, uric acid, urea, and malondialdehyde (MDA) levels and decrease in total protein, reduced glutathione (GSH), and catalase(CAT) compared to normal control animals, which indicates severe nephrotoxicity. Histopathological studies of kidney Gentamicin-treated animals showed extensive acute tubular necrosis and peri-tubular inflammation. Administration of EECC showed a significant improvement (p<0.05) in biochemical and oxidative stress markers compared to the disease group. EECC treated groups showed better histological appearance when compared to the disease group. Ethanolic extract of Carissa carandas Linn. Leaves showed significant nephroprotective activity against gentamicin-induced acute kidney injury.

N-Acetylcysteine Ameliorates Gentamicin-Induced Nephrotoxicity by Enhancing Autophagy and Reducing Oxidative Damage in Miniature Pigs.

ABSTRACTThe clinical use of gentamicin over prolonged periods is limited because of dose and time-dependent nephrotoxicity, in which intracellular oxidative stress and heightened inflammation have been implicated. Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. The aim of this study was to determine changes in autophagy in response to the antioxidant N-acetylcysteine (NAC), via its effects on oxidative stress, inflammation, apoptosis, and renal function, following treatment with gentamicin in mini pigs. Adult mini pigs were divided into isotonic saline solution, gentamicin, and gentamicin plus NAC combination treatment groups. Gentamicin-induced histopathological changes, including inflammatory cell infiltration and tubular necrosis, were attenuated by NAC. NAC ameliorated the gentamicin-induced decreases in the levels of autophagy-related proteins, such as LC3 (microtubule-associated protein 1 light chain 3), PINK1 (phosphatase and tensin homologue deleted on chromosome10-induced kinase 1), phospho-parkin, AMBRA1 (activatingmolecule in Beclin 1-regulated autophagy), p62/SQSTM1 (sequestosome protein 1), and polyubiquitinated protein aggregates. NAC also caused a significant reduction in oxidative damage markers, including 4-hydroxy-2-nonenal, protein carbonyls, γ-H2AX (gamma histone variant H2AX), and 8-hydroxy-2′-deoxyguanosine, in gentamicin-treated animals. These data show that the protective effects of NAC might be related, at least in part, to a reduced inflammatory response, as observed in animals treated with both gentamicin and NAC. These results suggest that autophagy could be a new therapeutic target for preventing gentamicin-induced kidney injury, and that NAC might ameliorate gentamicin-induced nephrotoxicity by autophagy.

Effective Treatment of Chronic Proliferative Cholangitis by Local Gentamicin Infusion in Rabbits.

Background Hepatolithiasis is highly prevalent in East Asia characterized by the presence of gallstones in the biliary ducts of the liver. Surgical resection is the potentially curative treatment but bears a high risk of stone recurrence and biliary restenosis. This is closely related to the universal presence of chronic proliferative cholangitis (CPC) in the majority of patients. Recent evidence has indicated the association of bacterial infection with the development of CPC in hepatolithiasis. Thus, this study aims to investigate the feasibility and efficacy of local infusion of gentamicin (an antibiotic) for the treatment of CPC in a rabbit model. Methods The rabbit CPC model was established based on previously published protocols. Bile duct samples were collected from gentamicin-treated or control animals for pathological and molecular characterization. Results Histologically, the hyperplasia of biliary epithelium and submucosal glands were inhibited and the thickness of the bile duct wall was significantly decreased after gentamicin therapy. Consistently, the percentage of proliferating cells marked by ki67 was significantly reduced by the treatment. More importantly, this treatment inhibited interleukin 2 production, an essential inflammatory cytokine, and the enzyme activity of endogenous β-Glucuronidase, a key factor in the formation of bile pigment. Conclusions Local gentamicin infusion effectively inhibits the inflammation, cell proliferation, and lithogenesis in a rabbit model of CPC. This approach represents a potential treatment for CPC and thus prevents recurrent hepatolithiasis.

Protective potential of Tamarindus indica against gentamicin-induced nephrotoxicity

Context: Gentamicin is an antibiotic that is effective against Gram-negative microorganisms. However, its clinical applications are often limited due to nephrotoxic effects.Objective: This study investigated the protective effects of aqueous-ethanol extract of Tamarindus indica L. (Leguminosae) fruits against gentamicin-induced renal toxicity.Materials and methods: A daily dose of 200 mg/kg of 70% aqueous-ethanol extract derived from T. indica was employed in male rabbits as a co-therapy with gentamicin (80 mg/kg) for a period of three weeks. Serum and urinary renal function parameters and histological assessments were carried out and compared with one way analysis of variance (Graphpad prism version 5.00, Graphpad Software, San Diego, CA).Results: The results showed that gentamicin-treated animals had significantly elevated blood urea nitrogen (54.1 ± 2.6 mg/dl), serum creatinine (4.0 ± 0.1 mg/dl), serum uric acid (2.3 ± 0.1 mg/dl) and urinary protein excretion (3.8 ± 0.3 mg/dl) with a fall in body weight (10 ± 1%), creatinine clearance (0.7 ± 0.09 ml/min), serum potassium (3.4 ± 0.1 mEq/l), serum calcium (7.6 ± 0.2 mg/dl), urinary volume (126 ± 9 ml/24 h) and urinary lactate dehydrogenase secretion (103.1 ± 4.2 U/l). However, animals treated by co-therapy with gentamicin and T. indica had significantly improved renal structure and function.Discussion and conclusion: Co-therapy of 200 mg/kg/d of T. indica for a period of three weeks successfully prevented functional and morphological derangements caused by gentamicin as assessed by different renal function parameters and histological examinations.

ALTERAÇÕES NO PESO FETAL, FÍGADO, PLACENTA E CORDÃO UMBILICAL DE RATAS INDUZIDAS PELA ADMINISTRAÇÃO DE GENTAMICINA DURANTE A PRENHEZ

SUMMARY Aminoglycosides are antibiotics used in the treatme nt of infections caused by gram-negative bacteria. They cross the placenta, are ototoxic in children and are nephroto xic in adults. The exposure to such antibiotics mus t be careful during fetal development. So, the purpose of this study wa s to verify their action in fetus and evaluate, mor phologically and morphometrically, the changes they induce in the fe tal liver, placental and umbilical cord. Albino Wis tar rats received a single intramuscular dose of 5 milligrams of gentam icin on the tenth day of pregnancy. The rats were s acrificed on the 20 th day. A delay in intra-uterine growth was macroscop ically observed. Gentamicin significantly reduced t he fetal weight, the placental weight and the length of umbi lical cord. Histologically, the gentamicin-treated animals exhibited livers with disorganized hepatocyte plaques and cap illary sinusoids. The hepatocytes showed a very vac uolated cytoplasm. Not withstanding the increased interstit ial connective tissue. It was concluded that the do ses of gentamicin used in this study caused no malformation, but indu ced changes in the fetal liver when administered du ring pregnancy.

P.1.g.077 Effects of clozapine on the prepulse inhibition of rats submitted to the model of schizophrenia induced by neonatal immune challenge

The aim of this study was to establish an animal model of acoustically evoked vestibulo-collic reflex using guinea pigs. A special clamp was applied to restrain the head and body of the guinea pigs, but leaving its four legs free. Each animal underwent vestibular evoked myogenic potential (VEMP) and caloric tests using clip electrode method without general anesthesia or decerebrate surgery. The response rates for the myogenic potentials on the neck of guinea pigs using 100, 90, 80 and 70 dB monaural acoustic stimulation with unilateral recording were 100%, 62%, 50% and 0%, respectively. The mean latencies of the positive and negative peaks for the myogenic potentials were 7.24 ± 0.49 and 9.15 ± 0.47 ms, 7.09 ± 0.43 and 9.28 ± 0.42 ms, as well as 7.03 ± 0.59 and 9.14 ± 0.56 ms, when elicited by 100, 90 and 80 dB acoustic stimulation, respectively. The median (minimum–maximum) peak-to-peak amplitudes were 11.93 (6.14–16.86), 10.99 (5.28–19.40), and 11.17 (5.02–20.72) μV, when elicited by 100, 90 and 80 dB acoustic stimulation, respectively. We found no significant relationship between the stimulus intensity and the mean latencies or peak-to-peak amplitude of the myogenic potentials in guinea pigs. For those treated with gentamicin unilaterally, all guinea pigs showed absent caloric responses on the lesion side, and absent myogenic potentials on the neck when using ipsi-lesional acoustic stimulation, while the hearing was preserved. Hence, the use of gentamicin-treated animals, along with normal controls and auditory brainstem responses, results in convincing results that the recorded myogenic potentials are in fact of vestibular origin.

Protective Effect of Cinnamomum tamala Extract on Gentamicin-Induced Nephrotic Damage in Rabbits

Purpose: To investigate the reno-protective properties of Cinnamomum tamala against gentamicininduced nephrotoxicity in rabbits.Methods: Rabbits were randomly divided into four groups (n = 6) including Group-1 (normal saline), Group-2 (gentamicin, 80 mg/kg/day), Group-3 (C. tamala, 200 mg/kg/day) and Group-4 (gentamicin, 80 mg/kg/day and C. tamala, 200 mg/kg/day). Body weight, blood urea nitrogen, serum creatinine, creatinine clearance, serum uric acid, urinary volume and urinary protein excretion were measured followed by histological examination.Results: Gentamicin-treated animals showed significant renal damage as indicated by rise in blood urea nitrogen (54.18 ± 2.60 mg/dl), serum creatinine (4.02 ± 0.14 mg/dl), serum uric acid (2.34 ± 0.12 mg/dl), urinary proteins (3.86 ± 0.32 mg/dl) and decrease in creatinine clearance (0.76 ± 0.09ml/min), urinary volume (126.00 ± 9.09 ml) and body weight (10.80 ± 1.09 %). However, animals treated with gentamicin and C. tamala significantly protected rabbit kidney from structural and functional changes associated with gentamicin.Conclusion: Based on the findings, it is apparent that concurrent administration of 200 mg/kg/day of C. tamala leaf extract and gentamicin effectively prevented gentamicin-induced renal damage.Keywords: Cinnamomum tamala, Renal protection, Gentamicin, Renal damage

Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.

Inhibition of cystathionine gamma-lyase and the biosynthesis of endogenous hydrogen sulphide ameliorates gentamicin-induced nephrotoxicity

Clinical use of gentamicin over prolonged periods is limited because of dose- and time-dependent nephrotoxicity. Primarily, lysosomal phospholipidosis, intracellular oxidative stress and heightened inflammation have been implicated. Hydrogen sulphide is an endogenously produced signal transduction molecule with strong anti-inflammatory, anti-apoptotic and cytoprotective properties. In several models of inflammatory disease however, tissue damage has been associated with increased activity of cystathionine gamma-lyase, biosynthesis of hydrogen sulphide and activation of leukocytes. The aim of this study was to determine effects of inhibiting hydrogen sulphide biosynthesis by DL-propargyl glycine (an irreversible inhibitor of cystathionine gamma-lyase) on inflammation, necrosis and renal function, following treatment with gentamicin in rats. Adult female Sprague–Dawley rats were divided into six groups and treated with; physiological saline, sodium hydrosulphide, DL-propargyl glycine, gentamicin, a combination of gentamicin and sodium hydrosulphide, or gentamicin and DL-propargyl glycine respectively. Gentamicin-induced histopathological changes including inflammatory cell infiltration and tubular necrosis were attenuated by co-administering gentamicin with DL-propargyl glycine (P<0.05 compared to saline controls and P<0.05 compared to gentamicin only). Similarly, DL-propargyl glycine caused a significant reduction (P<0.05) in lipid peroxidation, production of superoxide and the activation of tumour necrosis factor-alpha in gentamicin-treated animals. These data show that protective effects of DL-propargyl glycine might be related at least in part, to the reduced inflammatory responses observed in animals treated with both gentamicin and DL-propargyl glycine. Thus, enzyme systems involved in hydrogen sulphide biosynthesis may offer a viable therapeutic target in alleviating the nephrotoxic effects of gentamicin.

Restoration of 3D vestibular sensation in rhesus monkeys using a multichannel vestibular prosthesis

Profound bilateral loss of vestibular hair cell function can cause chronically disabling loss of balance and inability to maintain stable vision during head and body movements. We have previously shown that chinchillas rendered bilaterally vestibular-deficient via intratympanic administration of the ototoxic antibiotic gentamicin regain a more nearly normal 3-dimensional vestibulo-ocular reflex (3D VOR) when head motion information sensed by a head-mounted multichannel vestibular prosthesis (MVP) is encoded via rate-modulated pulsatile stimulation of vestibular nerve branches. Despite significant improvement versus the unaided condition, animals still exhibited some 3D VOR misalignment (i.e., the 3D axis of eye movement responses did not precisely align with the axis of head rotation), presumably due to current spread between a given ampullary nerve’s stimulating electrode(s) and afferent fibers in non-targeted branches of the vestibular nerve. Assuming that effects of current spread depend on relative orientation and separation between nerve branches, anatomic differences between chinchilla and human labyrinths may limit the extent to which results in chinchillas accurately predict MVP performance in humans. In this report, we describe the MVP-evoked 3D VOR measured in alert rhesus monkeys, which have labyrinths that are larger than chinchillas and temporal bone anatomy more similar to humans. Electrodes were implanted in five monkeys treated with intratympanic gentamicin to bilaterally ablate vestibular hair cell mechanosensitivity. Eye movements mediated by the 3D VOR were recorded during passive sinusoidal (0.2–5 Hz, peak 50°/s) and acceleration-step (1000°/s 2 to 150°/s) whole-body rotations in darkness about each semicircular canal axis. During constant 100 pulse/s stimulation (i.e., MVP powered ON but set to stimulate each ampullary nerve at a constant mean baseline rate not modulated by head motion), 3D VOR responses to head rotation exhibited profoundly low gain [(mean eye velocity amplitude)/(mean head velocity amplitude) < 0.1] and large misalignment between ideal and actual eye movements. In contrast, motion-modulated sinusoidal MVP stimuli elicited a 3D VOR with gain 0.4–0.7 and axis misalignment of 21–38°, and responses to high-acceleration transient head rotations exhibited gain and asymmetry closer to those of unilaterally gentamicin-treated animals (i.e., with one intact labyrinth) than to bilaterally gentamicin-treated animals without MVP stimulation. In comparison to responses observed under similar conditions in chinchillas, acute responses to MVP stimulation in rhesus macaque monkeys were slightly better aligned to the desired rotation axis. Responses during combined rotation and prosthetic stimulation were greater than when either stimulus was presented alone, suggesting that the central nervous system uses MVP input in the context of multisensory integration. Considering the similarity in temporal bone anatomy and VOR performance between rhesus monkeys and humans, these observations suggest that an MVP will likely restore a useful level of vestibular sensation and gaze stabilization in humans.

Doxycycline reduces nitric oxide production in guinea pig inner ears

Gentamicin application is an important therapeutic option to control vertigo spells in Ménière's disease. However, even in the case of low-dose intratympanic application, gentamicin might contribute to a pathological NO-increase leading to cochlear damage and hearing impairment. The study was performed to evaluate the nitric oxide (NO) reducing capacity of doxycycline in the inner ear after NO-induction by gentamicin. In a prospective animal study, a single dose of gentamicin (10mg/kg body weight) was injected intratympanically into male guinea pigs (n=48). The auditory brainstem responses (ABRs) were recorded prior to application and 3, 5 and 7 days afterwards. The organ of Corti and the lateral wall of 42 animals were isolated after 7 days and incubated separately for 6h in cell culture medium. Doxycycline was adjusted to organ cultures of 5 animals. Two NOS inhibitors, N(G)-Nitro-l-arginine methyl ester (l-NAME) and NG-monomethyl-l-arginine monoacetate (l-NMMA), were applied in three different concentrations to the organ cultures of 30 animals in total (5 animals per concentration). As controls, seven animals received no further substance except gentamicin. The NO-production was quantified by chemiluminescence. Additional six gentamicin-treated animals were used for immunohistochemical studies. The ABRs declined continuously from the first to the seventh day after gentamicin application. Doxycycline reduced NO-production in the lateral wall by 54% (p=.029) comparable to the effect of the applied nitric oxide inhibitors. In the organ of Corti, NO-production was reduced by about 41% showing no statistical significance in respect to great inter-animal variations. The application of doxycycline might offer a new therapeutic approach to prevent NO-induced cochlea damage through ototoxic substances.

Toxicoepigenetic Alteration of the Kidney Injury Molecule 1 Gene in Gentamicin-Exposed Rat Kidney

Chromatin modifications are now widely accepted as being essential steps involved in activation, repression, and poising of the expression of a large number of genes within the genome. Not only does understanding the role of such changes provide an opportunity to elucidate mechanisms controlling gene expression but in parallel offers the ability to develop novel indicative and predictive biomarkers of disease and toxicity. In the current study, we have applied the chromatin immunoprecipitation assay to investigate putative changes in the chromatin environment associated with the kidney injury molecule 1 (Kim1) gene upon exposure of rats to the nephrotoxicant, gentamicin. Chromatin was isolated from the kidneys of both control and gentamicin-treated animals and interrogated using specific antibodies recognizing two modifications of histone H3, acetylation of lysine 9, and trimethylation of lysine 4, along with RNA polymerase II. Enriched chromatin fractions were analyzed by quantitative PCR using tiled primer pairs covering 4 kb of the Kim1 gene (spanning -2 kb to + 2 kb, relative to the transcription start site). The results demonstrate a substantial increase in the presence of RNA polymerase II and both histone modifications following gentamicin treatment in regions downstream but not upstream of the transcriptional start site of the Kim1 gene. These changes were associated with a marked increase in messenger RNA coding for the Kim1 protein. Together these data suggest, for the first time, that the Kim1 gene is regulated in an epigenetic fashion under conditions of nephrotoxicity.

Assessment of gentamicin-induced vestibulotoxicity by click and galvanic vestibular-evoked myogenic potentials: A guinea pig investigation

Objective The aim of this investigation carried out with guinea pigs was to study the possible effects of a gentamicin treatment on the saccular macula and on its afferent vestibular ganglion neurons. Methods The gentamicin-induced impairment was analyzed using vestibular-evoked myogenic potentials (VEMPs) elicited by both click and galvanic vestibular stimulations (GVS). Fifty μl of saline or gentamicin solution (40 mg/ml) was dropped over the round window membrane of the right (control) and left (lesion) cochleae, respectively. Four weeks after surgery, the VEMPs elicited with clicks and GVS were evaluated for each animal. Then, the animals were sacrificed in order to perform morphological and anti-Nav1.8 immunocytochemical analyses. Results Click- and GVS-VEMPs were obtained in all of the controls, whereas no potentials were obtained from gentamicin-treated animals. Lesions of sensory cells were observed in the saccular macula. In the injured vestibular ganglion, the percentage of voltage-gated sodium channel Nav1.8-like immunoreactive (Nav1.8-LI) neurons was significantly lower (38.9 ± 0.7) than that (53.6 ± 3.2) calculated in controls. Conclusions Gentamicin-induced impairments of the saccular macula and afferents of guinea pigs can be evaluated by recording both click- and GVS-VEMPs. Both tests provide information on the sacculo-collic reflex pathway and could help a clinical diagnosis of gentamicin intoxication by conventional eardrops in the patient with a perforated eardrum.

Comparative Analysis of Novel Noninvasive Renal Biomarkers and Metabonomic Changes in a Rat Model of Gentamicin Nephrotoxicity

Although early detection of toxicant induced kidney injury during drug development and chemical safety testing is still limited by the lack of sensitive and reliable biomarkers of nephrotoxicity, omics technologies have brought enormous opportunities for improved detection of toxicity and biomarker discovery. Thus, transcription profiling has led to the identification of several candidate kidney biomarkers such as kidney injury molecule (Kim-1), clusterin, lipocalin-2, and tissue inhibitor of metalloproteinase 1 (Timp-1), and metabonomic analysis of urine is increasingly used to indicate biochemical perturbations due to renal toxicity. This study was designed to assess the value of a combined (1)H-NMR and gas chromatography-mass spectrometry (GC-MS) metabonomics approach and a set of novel urinary protein markers for early detection of nephrotoxicity following treatment of male Wistar rats with gentamicin (60 and 120 mg/kg bw, s.c.) for 7 days. Time- and dose-dependent separation of gentamicin-treated animals from controls was observed by principal component analysis of (1)H-NMR and GC-MS data. The major metabolic alterations responsible for group separation were linked to the gut microflora, thus related to the pharmacology of the drug, and increased glucose in urine of gentamicin-treated animals, consistent with damage to the S(1) and S(2) proximal tubules, the primary sites for glucose reabsorption. Altered excretion of urinary protein biomarkers Kim-1 and lipocalin-2, but not Timp-1 and clusterin, was detected before marked changes in clinical chemistry parameters were evident. The early increase in urine, which correlated with enhanced gene and protein expression at the site of injury, provides further support for lipocalin-2 and Kim-1 as sensitive, noninvasive biomarkers of nephrotoxicity.

Ototoxicity on cochlear nucleus neurons following systemic application of gentamicin

Conclusion. The gentamicin-induced pathological alteration in the cochlear nucleus is not exclusively a secondary consequence of the damage in the cochlea. Instead, the toxic effect of gentamicin on the cochlear nucleus may occur simultaneously or even earlier than that on the cochlea. Objectives. To investigate the pathological alteration of cochlear nucleus neurons in guinea pigs following systemic application of gentamicin. Materials and methods. Guinea pigs were injected with gentamicin for 1 day, 3 days, 1 week, 2 weeks, and 3 weeks, respectively. In gentamicin-treated animals, the hearing function was evaluated by measuring the auditory brainstem response (ABR). The number and cross-sectional area of substance P-positive neurons in the cochlear nucleus were also measured. Results. The threshold of ABR and the number of substance P-positive neurons in the cochlear nucleus were significantly increased after 1 week and 3 days of injection of gentamicin, respectively. The cross-sectional area of substance P-positive neurons in the cochlear nucleus was significantly reduced after 1-day injection of gentamicin.

Protective effect of melatonin against gentamicin ototoxicity

To research the protective effect of melatonin against gentamicin ototoxicity. Guinea pigs were randomly divided into four groups. The first group received intramuscular gentamicin (120 mg/kg body weight/day) for 17 days. Over the same time period, a second group simultaneously received intramuscular gentamicin (120 mg/kg body weight/day) plus (on the other side) intramuscular melatonin (0.3 ml kg body weight/day). Two groups of controls were treated for 17 days with either intramuscular melatonin or intramuscular saline. After the 17 days, each animal underwent distortion product otoacoustic emission testing (both ears). The guinea pigs were sacrificed by decapitation just after the final injection. Their cochleae were used to produce a tissue section, surface preparation and scanning electron microscope preparation. Distortion product otoacoustic emission testing indicated gentamicin-induced hearing loss at 3, 4, 6 and 8 kHz in gentamicin-treated animals. Animals receiving melatonin co-therapy had significantly attenuated hearing loss and their cochleae showed lower rates of outer hair cell loss (comparing the same cochlear turns), compared with gentamicin-treated animals (p < 0.01). These findings confirm the occurrence of outer hair cell loss after gentamicin treatment, and the attenuation of such loss following simultaneous melatonin injection, using the method of morphological evaluation. These results suggest that melatonin protects against gentamicin ototoxicity by interfering with cytotoxic mechanisms.

Vestibular Evoked Myogenic Potentials Are Heavily Dependent on Type I Hair Cell Activity of the Saccular Macula in Guinea Pigs

This study applied the vestibular evoked myogenic potential (VEMP) test to guinea pigs coupled with electronic microscopic examination to determine whether VEMPs are dependent on type I or II hair cell activity of the saccular macula. An amount of 0.05 ml of gentamicin (40 mg/ml) was injected directly overlaying, but not through, the round window membrane of the left ear in guinea pigs.One week after surgery, auditory brainstem response test revealed normal responses in 12 animals (80%), and elevated thresholds in 3 animals (20%). The VEMP test using click stimulation showed absent responses in all 15 animals (100%). Another 6 gentamicin-treated animals underwent the VEMP test using galvanic stimulation and all 6 also displayed absent responses. Ultrathin sections of the saccular macula in the gentamicin-treated ears displayed morphologic alterations in type I or II hair cells, including shrinkage and/or vacuolization in the cytoplasm, increased electron density of the cytoplasm and nuclear chromatin, and cellular lucency. However, extrusion degeneration was rare and only present in type II hair cells. Quantitative analysis demonstrated that the histological density of intact type I hair cells was 1.1 ± 1.2/4000 μm² in the gentamicin-treated ears, showing significantly less than that in control ears (4.5 ± 1.8/4000 μm²). However, no significant difference was observed in the densities of intact type II hair cells and supporting cells between treated and control ears. Furthermore, the calyx terminals surrounding the damaged type I hair cells were swollen and disrupted, while the button afferents contacting the damaged type II hair cells were not obviously deformed. Based on the above results, we therefore conclude that VEMPs are heavily dependent on type I hair cell activity of the saccular macula in guinea pigs.

Glomerular basement membrane alterations induced by gentamicin administration in rats

The widespread therapeutic use of the aminoglycoside antibiotic gentamicin (GM) is limited by its nephrotoxic side effect, which can lead to acute renal failure. This study aimed at examining effects of high, supratherapeutic doses of gentamicin on morphological, structural and functional alterations of the glomerular basement membrane in adult rats. Experiments were done on 30 male Wistar rats, divided into two experimental groups. GM-group (20 rats) received gentamicin at a dose of 100 mg/kg intraperitoneally during eight consecutive days. Control or C-group (10 rats) received 1 ml/day saline intraperitoneally. For histological analysis, 5 μm thick sections were stained with hematoxylin and eosin (HE), periodic acid Schiff (PAS), and Jones methenamine silver. Glomerular basement membrane thickness, glomerular area, major and minor axes, perimeter, diameter, roundness and mean optical density were analyzed. Biochemical analyses were used to determine concentrations of blood urea, serum creatinine, sodium and potassium. In GM-group rats, glomeruli were larger and glomerular basement membrane was diffusely and irregularly thickened with neutrophil cell infiltration. Glomerular area, major axis, minor axis, diameter and perimeter were significantly higher in GM-group compared to C-group rats. Opposite to this, glomerular optical density and average roundness were larger in C-group than in gentamicin-treated animals. Our results clearly showed morphological and structural alterations of glomeruli and glomerular basement membrane as well as alterations of proximal tubules in adult rats exposed to high doses of gentamicin.

Effects of medial olivocochlear efferent stimulation on the activity of neurons in the auditory midbrain

Medial olivocochlear (MOC) efferents are known to suppress spontaneous activity and sound-evoked responses of primary afferents by their actions on outer hair cells in the cochlea. This study investigated the effects of MOC activation on the responses of single neurons in the central nucleus of the inferior colliculus (CNIC) of anaesthetized guinea pigs. Extracellular responses of CNIC neurons to contralateral tones were recorded with and without MOC stimulation in normal animals and in animals acutely treated with gentamicin to eliminate peripheral effects of MOC activation. In normal animals, input-output functions of CNIC neurons showed a variety of changes. Some effects resembled qualitatively those reported for primary afferents. However, other effects were also observed, including an increase of firing rates at medium- to high-tone levels and in a small number of neurons (10%), an increase in spontaneous activity. In addition, larger threshold shifts and larger reductions of spontaneous firing rates were observed as compared to effects seen in the periphery. In gentamicin-treated animals, activation of MOC efferents did not produce any changes in the input-output functions or spontaneous activity of CNIC neurons. This observation is consistent with the majority of MOC-induced changes in monaural responses in the CNIC being mediated by the actions of MOC terminals in the cochlea and resulting from the interplay between altered afferent input and central circuitry.