Establishment of a Gentamicin Cochlear Poisoning Model in Guinea Pigs and Cochlear Nerve Endings Recognition of Ultrasound Signals.

BACKGROUND Aminoglycosides, a type of gram-negative antibacterial, are broad-spectrum antibiotics that are highly potent and have satisfactory therapeutic efficacy in the treatment of life-threatening infections. Our study aimed to establish a gentamicin-induced cochlear injury model and to investigate the cochlear nerve endings' recognition of ultrasound signals. MATERIAL AND METHODS A guinea pig cochlear injury model was established by intraperitoneal injection of gentamycin. Auditory brainstem response (ABR) and fMRI an affected cerebral cortex region of interest (ROI) of the cerebral cortex blood oxygenation level dependent (BOLD) effect was induced by bone-conducted ultrasound. Immunofluorescence was used to detect expression of Prestin in outer hair cells, Otoferlin in inner hair cells, and cochlear hair cell microfilament protein (F-Actin). RESULTS For 30-35 KHz bone-conducted ultrasound, the induction rate of ABR threshold or ROI in the control group and the cochlear injury group was 40% and 0%, respectively, and for 80-90 KHz the induction rate was 20% and 20%, respectively. Gentamicin poisoning induced downregulation of expression of Prestin in cochlear outer cochlea, and Otoferlin and F-Actin in cochlear hair cells in different regions. CONCLUSIONS Gentamicin poisoning can cause different degrees of damage to cochlea hair cells in different regions. Guinea pigs with gentamicin poisoning can recognize high-frequency ultrasonic signals.

[Gentamicin intoxication in a preterm infant]. / Gentamicin Intoxikation bei einem Frühgeborenen.

Gentamicin is an aminoglycoside that is widely used in neonatology in spite of its known nephrotoxicity and ototoxicity. Because there are only few cases reported in the literature experience with gentamicin overdosage is limited. We report the case of a preterm (gestational age 32+2 weeks) infant with an accidental administration of a ten-fold dose of gentamicin. The baby was treated with a slight increase of fluid intake and monitoring of renal function and gentamicin levels, respectively. A rapid decrease of the gentamicin level (peak level 44.5 mg/L, extrapolated peak level 65 mg/L) was observed. Nephrotoxicity or ototoxicity did not occur. Because of the small number of described cases, a general recommendation for the management of gentamicin intoxication is not possible. The intensity of treatment depends on renal function and gentamicin level. Only isolated patients will need dialysis or exchange transfusion. The case also demonstrates the need for the continuous discussion about hospital-associated damage and error management systems.

Effect of intratympanic application of aminoglycosides on click-evoked myogenic potentials in Guinea pigs.

OBJECTIVES: Although numerous studies have identified damage to the cochlear and vestibular end organs as the primary site of aminoglycoside-induced ototoxicity, the effect on the saccule remains poorly understood, possibly due to lack of monitoring saccular function in experimental animals. Therefore, this study applied three kinds of aminoglycosides into the tympanic space of guinea pigs to examine their toxic impact on the saccule by way of click-evoked myogenic potential test coupled with morphologic assessment. DESIGN: Albino guinea pigs were treated with saline, gentamicin, tobramycin, or amikacin, with 10 animals assigned to each group. Each compound was injected directly overlying but not through the round window membrane on the left ear, with the right ear serving as a control. One week after injection, each animal underwent auditory brain stem response, caloric test, and click-evoked myogenic potential test. Animals were then killed for morphologic assessment through the use of light and electron microscopic examinations. RESULTS: The animals treated with saline, gentamicin, tobramycin, or amikacin exhibited abnormal auditory brain stem response in 0%, 30%, 100%, and 30% of cases; abnormal caloric responses were found in 0%, 100%, 40%, and 40% of cases; absent click-evoked myogenic potentials were found in 0%, 100%, 30%, and 40% of cases, respectively. Gentamicin and other groups differed significantly in abnormal rates of caloric responses and click-evoked myogenic potentials. Morphologic study of the gentamicin-treated animals confirmed that the absence of click-evoked myogenic potential originated from the lesion in the saccular macula. CONCLUSIONS: Gentamicin represents the dominant susceptibility of aminoglycoside-induced vestibulotoxicity for eliminating both semicircular canal and saccular functions. This study further confirms the findings of human studies in which the caloric and vestibular evoked myogenic potentials responses were monitored to assess the abolition of vestibular function in patients treated with intratympanic gentamicin injection.

Altered NMDA receptor expression in renal toxicity: Protection with a receptor antagonist.

BACKGROUND: The N-methyl-d-aspartate (NMDA) receptor is expressed in the kidney. The receptor plays a major role in gentamicin ototoxicity. We assessed the role of the renal NMDA receptor subunits NR1 and NR2C in a model of gentamicin nephrotoxicity. METHODS: Rats were exposed to either saline (control), high-dose, short-term gentamicin, or short-term gentamicin plus the NMDA antagonist MK-801 (short-term gentamicin + MK-801) for 3 days. RESULTS: Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed that NR1 mRNA expression was significantly higher (P= 0.03) in the renal cortex of short-term gentamicin rats. NR2C subunit mRNA expression was unaltered in short-term gentamicin rats. Western blot analysis revealed that NR1 (P= 0.009) and NR2C (P= 0.003) protein abundance was significantly higher in the renal cortex short-term gentamicin rats. We assessed two potential intracellular pathways that may mediate short-term gentamicin/NMDA. Calpain I and II expression was similar in short-term gentamicin and control rats. Endothelin type B receptor (ETBR) expression was significantly increased in the renal cortex of short-term gentamicin rats (P= 0.0003), and urinary nitrite concentration (reflecting nitric oxide) was significantly increased in short-term gentamicin rats (P= 0.03). Serum creatinine was significantly elevated in short-term gentamicin animals (P= 0.03), and this increase was attenuated in short-term gentamicin + MK-801 rats. Blood pressure was higher in short-term gentamicin rats; this was attenuated in short-term gentamicin + MK-801 rats. Urine pH was significantly lower in short-term gentamicin (P < 0.0001) rats; this was reversed in short-term gentamicin + MK-801 (P= 0.005) rats. Urinary nitrite was significantly higher in short-term gentamicin rats; this was normalized in short-term gentamicin + MK-801 rats. MK-801 alone had no effect on clinical parameters. CONCLUSION: NMDA receptor subunit expression is increased in short-term gentamicin animals, and the receptor likely mediates cell damage via the endothelin-ETBR-nitric oxide pathway. NMDA antagonism ameliorated renal damage after exposure to short-term gentamicin.

Late dosing with ethacrynic acid can reduce gentamicin concentration in perilymph and protect cochlear hair cells.

A key factor in the well-known interaction between ethacrynic acid (EA) and aminoglycoside antibiotics (AABs) is disruption of the blood-labyrinth barrier (BLB), leading to rapid entry of EA and AABs into the cochlear fluids. The idea that the blood-labyrinthine fluid concentration gradient might be utilized in a protective manner was tested in the current experiment. We hypothesized that administering EA when gentamicin (GM) levels are higher in the cochlea than in the blood might actually reduce cochlear damage by permitting efflux of GM from the cochlear fluids into the bloodstream, down a concentration gradient and across a temporarily disrupted BLB. Guinea pigs received 1, 11, 14 or 20 injections of GM (125 mg/kg i.m.). Approximately half of the animals also received a single injection of EA (40 mg/kg i.v.) either concurrently or 12-18 h after the last GM injection. Concurrent injection of EA significantly increased GM concentration in serum and perilymph at all time points sampled (2.5, 5-8, and 12 h post injection). Compared to animals that received GM only, animals that received a delayed injection of EA had a significantly lower GM concentration in perilymph, lower thresholds of the compound action potential, and less outer hair cell loss. Collectively, the evidence suggests that EA can reduce GM ototoxicity if it is administered 12-18 h after GM, but the mechanism remains to be elucidated. The results may have implications for the clinical management of aminoglycoside ototoxicity in humans, as well as for understanding the mechanisms underlying AAB/EA interactions.

Comparative vestibulotoxicity of different aminoglycosides in the Guinea pigs.

The histopathological alterations in the vestibule due to aminoglycosides are well defined. Although there are reports comparing the vestibulotoxic effects of the many aminoglycosides, this is the first study to compare the effects of the most commonly used aminoglycosides i.e., streptomycin, gentamicin, amikacin and netilmicin administered both transtympanically and systemically. The transtympanic and systemic administration of each aminoglycoside caused similar histopathological alterations in the vestibule. The most severe degeneration in the cristae ampullaris, utriculus and sacculus was observed after streptomycine administration. The severity of the vestibular damage in terms of magnitude was in the order of streptomycine, gentamicin, amikacin, and netilmicin.

Dietary composition alters gentamicin-induced nephrotoxicity in rats.

Previous studies have shown temporal variations in gentamicin-induced renal toxicity characterized by a peak when administered during the resting period and a trough during the active period. This time-dependent toxicity was also altered according to the macronutrient composition of dietary regimens offered to female rats. In the present study, adult female Sprague-Dawley rats were adapted to semipurified isocaloric diets containing 20% casein or soy-protein (10% fat each) or to a standard chow diet (18.1% mixed proteins; 4.5% fat). The animals were then chronically treated for 10 days with a nephrotoxic dose of gentamicin sulfate (40 mg/kg/day ip) or a saline solution administered in the middle of their resting period (1200 h) or in the middle of their activity period (0000 h). Body weights of rats injected in the middle of their resting period decreased over the last 6 days of gentamicin treatment. Total 12-h light and 12-h dark food intakes were decreased in gentamicin-treated rats. Rats fed the standard chow diet had significantly lower corticocellular regeneration, serum creatinine and blood urea nitrogen compared to those fed the casein- and soy-containing diets. The present study demonstrates that chronic gentamicin-induced renal toxicity varies temporally according to the time of administration and that a mixed protein diet containing a lower fat level can protect against gentamicin-induced nephrotoxicity.

Anatomic and morphometric changes to gerbil posterior cristas following transtympanic administration of gentamicin and streptomycin.

Many studies have sought to document ototoxic damage and to study repair and regeneration of mammalian vestibular sensory epithelia. However, linear density analysis of the sensory cells or use of methods that focus on detection of actin in the stereocilia and cuticular plates at the reticular lamina detect only the disappearance of "hair cells" as defined by a narrow set of criteria. The research presented here focuses on the effects of two ototoxic drugs (gentamicin and streptomycin). We used light microscopic analysis of semithin sections to observe changes in the distribution of sensory and supporting cell nuclei and to elucidate other, previously undetected, morphological changes that occurred within the vestibular epithelia. Age-matched untreated and vehicle-treated controls showed that the gerbil posterior crista is asymmetrical on either side of the septum cruciatum; the longer end is taller and narrower than the shorter end. In cross sections taken throughout the length of each posterior crista, the thickness of the sensory epithelium along the sides (peripheral zone) is greater than at the apex (central zone). In tissue sections of the sensory epithelium, the ratio of sensory cell nuclei to support cell nuclei is slightly over 1:1.5 in all regions except the septum cruciatum where most sensory cells are absent and supporting cells predominate. In tissue sections from the most damaged drug-treated specimens, there was a decrease in the linear density of nuclei in the sensory cell layer, with a compensatory increase in the linear density of nuclei in the support cell layer of the sensory epithelia. In these specimens, linear density of total nuclei/tissue section remained the same. In these regions, the width of the epithelium became up to 50% thinner. The ratio of sensory to supporting cell nuclei changed to 1:6. Drug exposure led additionally to a decrease in length of the cristas, but there was not a linear relationship between the change in length of the crista and length of the septum cruciatum in these shorter cristas. In drug-treated cristas, other changes included a decrease in calculated surface area and volume of the epithelia. Thus, while linear density measurements of sensory cell nuclei provide an indication of damage, there are additional anatomic changes to the cristas and caution is advised with regard to interpreting changes as "loss" of cells.

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.

Hair cell differentiation in chick cochlear epithelium after aminoglycoside toxicity: in vivo and in vitro observations.

Inner ear epithelia of mature birds regenerate hair cells after ototoxic or acoustic insult. The lack of markers that selectively label cells in regenerating epithelia and of culture systems composed primarily of progenitor cells has hampered the identification of cellular and molecular interactions that regulate hair cell regeneration. In control basilar papillae, we identified two markers that selectively label hair cells (calmodulin and TUJ1 beta tubulin antibodies) and one marker unique for support cells (cytokeratin antibodies). Examination of regenerating epithelia demonstrated that calmodulin and beta tubulin are also expressed in early differentiating hair cells, and cytokeratins are retained in proliferative support cells. Enzymatic and mechanical methods were used to isolate sensory epithelia from mature chick basilar papillae, and epithelia were cultured in different conditions. In control cultures, hair cells are morphologically stable for up to 6 d, because calmodulin immunoreactivity and phalloidin labeling of filamentous actin are retained. The addition of an ototoxic antibiotic to cultures, however, causes complete hair cell loss by 2 d in vitro and generates cultures composed of calmodulin-negative, cytokeratin-positive support cells. These cells are highly proliferative for the first 2-7 d after plating, but stop dividing by 9 d. Calmodulin- or TUJ1-positive cells reemerge in cultures treated with antibiotic for 5 d and maintained for an additional 5 d without antibiotic. A subset of calmodulin-positive cells was also labeled with BrdU when it was continuously present in cultures, suggesting that some cells generated in culture begin to differentiate into hair cells.

[Effects of the calcium-channel blocker diltiazem on gentamicin-induced nephrotoxicity in rats]. / Effets d'un inhibiteur des canaux calciques, le diltiazem sur la néphrotoxicité de la gentamicine chez le rat.

Injection of diltiazem (40 mg/kg/d) to gentamicin (75 mg/kg/d = G 75 or 100 mg/kg/d = G 100) treated rats enhances aminoglycoside-induced nephrotoxicity. As a result of this combination, acute renal failure becomes systematic and is often irreversible. The lesion is of tubular origin and is characterized by a large increase in the urinary N-acetyl-beta-D-glucosaminidase (u-NAG) activity and its NAG-B isoenzyme level. The phenomenon is twice as marked with G 75 (u-NAG x 6.8, NAG-B x 2.2) as with G 100 (u-NAG x 3.1, NAG-B x 1.1). The effect seems to be attenuated if diltiazem is administered as a preventive treatment or in drinking water. As well as its diuretic properties, diltiazem may aggravate the renal toxicity of gentamicin by reducing the proximal tubular availability of calcium. Diltiazem inhibits reabsorption and behaves like a non-competitive inhibitor of calcium. This deficiency favours the proximal tubular binding and the non-specific penetration of gentamicin in the cytosol and cellular organelles (microsomes, mitochondria). The tubular toxic symptoms which ensure (inactivation of membranaceous enzyme, reduction of microsomal protein synthesis and ATP level, decreased of solute reabsorptive flux) lead in turn to proximal tubular necrosis and acute renal failure.

Retinal toxicity of intravitreal gentamicin.

The short- and long-term effects of a 10-mg dose of intravitreal gentamicin were studied in the subhuman primate eye with regard to the changes in clinical appearance, fluorescein angiography, electroretinography, histopathologic finding, and electron microscopy. The gentamicin produced retinal whitening with a cherry-red spot, generalized vascular incompetence, diffuse retinal necrosis, thrombosis of the large retinal blood vessels, widespread loss of the retinal capillary pericytes and endothelial cells, and a rapid extinction of the electroretinogram.

Reduction of the drug-induced nephrotoxicity by ATP-MgCl2. II. Effects on gentamicin-treated isolated perfused kidneys.

Drug-induced nephrotoxicity (NT) has become an increasingly significant clinical problem. An in vitro model of drug-induced NT was therefore developed using gentamicin and the effects of ATP-MgCl2 on reduction or prevention of NT were determined. To study this, non-pulsatile perfusion in isolated rat kidneys was maintained at 100 mm Hg during 2 hr of perfusion at 37 degrees C. The oxygenated Krebs-HCO3 perfusate contained 7.5 g/dl albumin as colloid, glucose, creatinine, amino acids, trace amounts of [3H]inulin and 125I-lysozyme, and either 0, 0.4, 0.8, or 1.2 mg/ml of gentamicin. In some studies, 2 mM ATP-MgCl2 was added with 0.8 mg/ml of gentamicin at 0 and 60 min of perfusion. During each 10-min clearance period, glomerular filtration rates, sodium absorption, water absorption, and fractional clearance of TCA-precipitable lysozyme were measured. The results indicate that renal perfusate flow, glomerular filtration rate, urinary flow and tubular absorption of protein (a sensitive indicator of tubular function), sodium, and water were affected by gentamicin in a dose-dependent manner. An isolated kidney preparation can therefore be used to study gentamicin-induced NT. Higher in vitro perfusate concentrations of the drug were needed, however, to acutely mimic the in vivo cumulative effects. Nonetheless, renal perfusate flow, glomerular filtration rate, and the depression in protein reabsorption which occurred with gentamicin treatment were markedly improved by simultaneous treatment with ATP-MgCl2. Thus, ATP-MgCl2 may be useful in reducing drug-induced nephrotoxicity.

Incidence of potentially toxic concentrations of gentamicin in the neonate.

The incidence of putatively toxic serum concentrations and the factors influencing their occurrence were investigated in a study of 91 neonates receiving parenteral gentamicin twice daily at a dose of mean (SD) 5.5 (0.1) mg/kg/day. Most neonates were preterm and of low birthweight. Serum concentrations, area under the curve (AUC), and clearance were calculated. Potentially toxic trough concentrations (greater than 2 mg/l) were recorded in 57 of 91 (63%) neonates; 24 of these had trough concentrations greater than 3 mg/l. These babies were of a significantly lower gestational age and were younger than the remainder of the population. Toxic trough concentrations were not accompanied by raised peak serum values. A wide variation in all pharmacokinetic variables was observed. Peak serum concentration was most highly correlated with dose, while trough concentration, AUC, and clearance were more dependent on postnatal age. Clearance of gentamicin decreased significantly with increasing serum urea and creatinine concentrations. Preterm neonates in the first week of life are likely to develop potentially toxic serum concentrations when receiving the currently recommended dose of gentamicin (5-6 mg/kg/day). To prevent accumulation the dosage interval may need to be increased to 18 hours in these babies.

Gentamicin extraction from an anuric patient by combined haemodialysis and charcoal haemoperfusion.

A 39-year-old woman who developed acute renal failure following intra-abdominal sepsis was treated with gentamicin. Her serum concentrations reached potentially toxic levels. Combined haemoperfusion and haemodialysis removed approximately 70% of the given drug and the patient made a complete recovery.