Experimental observation on cochlea toxicity of kanamycin combined with furosemide in guinea pigs

Objective To investigate the toxic effect of kanamycin combined with furosemide on guinea pig cochlea. Methods 25 guinea pigs in the experimental group were injected with kanamycin 500 mg/kg intramuscularly and furosemide 50 mg/kg intravenously 2 hours later, while 4 guinea pigs in the control group. On the 7th day after administration, auditory brainstem evoked potentials were used to detect the auditory function of guinea pigs in the experimental group and the control group. The cochlea of guinea pigs with threshold greater than 95 dB SPL was observed by immunofluorescence staining and routine sectioning, and the cochlea was observed by scanning electron microscope. Results the ABR test threshold of experimental group 13 guinea pigs was higher than 95 dB SPL. Observation on deaf guinea pigs showed that hair cells and nerve fibers were obviously damaged, especially the first and second cochlea. Conclusion Kanamycin combined with furosemide can cause serious damage to hair cells and nerve fibers in guinea pigs, which is a rapid and effective method to establish deafness model.

Kanamycin; Furosemide; Auditory brainstem evoked potential; Histopathology of cochlea; immunofluorescence

Objective to investigate the ototoxicity of kanamycin combined with loop diuretic furosemide in guinea pigs Methods Guinea pigs were injected with KM (500 mg/kg) intramuscularly and furosemide (50 mg/ kg) intravenously 2 hours later. Auditory brainstem response (ABRs) was recorded on the 7th day after administration to monitor the hearing of animals. Immunohistochemical and histopathological changes were observed by light microscope and scanning electron microscope. Results The subsequent ABR monitoring showed that the deep hearing loss was bilateral and permanent. Histopathological examination showed that the inner and outer hair cells of the basal cochlea were missing. The degree of nerve fiber injury also exists in the basilar cochlea and depends on the survival time after deafening surgery. Conclusion the combination of KA and furosemide can effectively cause deep hearing loss in guinea pigs, and it is an effective method to cause deafness in acute animal experiments.

Keywords kanamycin (km); Furosemide; Auditory brainstem response; ; Histopathology of cochlea

The advantage of establishing animal deafness model by intramuscular injection of kanamycin (KM) and uric acid (EA) is that it can cause deafness without stimulating cochlea or round window [1]. Kanamycin is an aminoglycoside antibiotic, and its clinical and experimental studies on inner ear toxicity have been reported. Furosemide is a loop diuretic. The experimental study of furosemide toxicity shows that furosemide can cause pathological changes of marginal cells of stria vascularis. Our previous experiments used auditory brainstem response and other tests to prove that the auditory function of guinea pigs was seriously damaged three days after the combination of KM and ea [3]. In this study, frozen sections, succinate dehydrogenase (SDH) staining, immunofluorescence staining and scanning electron microscopy were used to evaluate the toxic effects of kanamycin combined with furosemide on guinea pig cochlea from the perspective of inner ear pathomorphology.

1 materials and methods

1. 1 animal grouping

Twenty-nine healthy adult white red-eye guinea pigs, with sensitive auricle response, of either sex, weighing 250-300 g (provided by the Experimental Animal Center of the General Hospital of PLA), were randomly divided into two groups, 25 in the experimental group and 4 in the blank control group.

1.2 Animal medication

In the experimental group, the guinea pigs were injected with kanamycin sulfate 500 mg/kg once, and then injected with furosemide 2 hours later: the anesthetized animals were injected with Sumianxin 0.0 1 ml /kg to expose 1 jugular vein during the operation, and injected with furosemide 50 mg/kg within 30 seconds [3].

Kanamycin sulfate: 250,000 units /ml, produced by Tianjin Pharmaceutical Jiaozuo Co., Ltd., batch number: 06051531; Furosemide: 10 mg/ml, produced by Tianjin Jinyao Amino Acid Co., Ltd., batch number: 0606 19 1. Su Mianxin: 1.5 ml, provided by Institute of Animal Husbandry of PLA Quartermaster University. Its main components are haloperidol, xinmianling and ketamine.

1.3 auditory brainstem response threshold test

After 7 days, the two groups of guinea pigs were tested for bilateral ABR threshold in the sound-proof shielding room with Smart EP 2.22 system, an intelligent hearing system of American Zhiting Company. The stimulus sound uses a burst tone, the band-pass filtering width is 300 ~ 3 000 Hz, the superposition times are 65438 0024 times, and the scanning time is 65438 00 ms. The electrodes are arranged as follows: the skull top is the recording electrode, the ear is the reference electrode, and the grounding electrode is placed at the tip of the nose. Short pure tones of 2 kHz, 4 kHz, 8 kHz and 16 kHz are used as stimulus tones.

1.4 sample preparation

After decapitation, take out the temporal bone, open the auditory vesicle, drill a small hole at the top of the cochlea, and open the oval window and the round window at the same time; Then 4% paraformaldehyde fixative was poured into the cochlea from the small hole at the tip of the cochlea with a straw until the liquid flowed out of the two windows, and then the temporal bone was immersed in the fixative for fixation. Eight guinea-pig cochlea with ABR threshold greater than 95 dB SPL in the experimental group and three cochlea in the control group were taken for routine frozen section, four guinea-pig cochlea with ABR threshold greater than 95 dB SPL in the experimental group and three cochlea in the control group were stained with immunofluorescence, and four guinea-pig cochlea with ABR threshold greater than 95 dB SPL in the experimental group and two guinea-pig cochlea in the control group were taken to prepare scanning electron microscope samples.

1.5 immunohistochemical staining

Cochlear specimens were washed with 0.0 1 mol/L phosphate buffer (PBS) for three times. 0. 1% Triton-PBS for three minutes. 10% sheep serum (diluted in 0.0 1% Triton-PBS) was sealed at room temperature for 30 minutes, and the sheep serum sealing liquid was poured out without washing. Adding rabbit-derived myosin antibody and mouse-derived neurofilament antibody (SIGMA Biotechnology Company), diluting with 3% sheep serum Triton-PBS, and freezing at 4℃ overnight. Wash with 0. 1% Triton-PBS 10 minutes, three times each time. Add secondary antibody (goat anti-rabbit and goat anti-mouse IgG antibody labeled with fluorescent dye Alexa Flour 488) and incubate at room temperature in the dark for one hour. PBS wash 10 minutes three times each. Anti-quenching sealing sheet. Observed by laser scanning * * * focusing microscope (LSM Zeiss 5 10 Meta, Germany).

1.6 sample preparation for scanning electron microscope

After cochlear tissue was taken out, all specimens were fixed with glutaraldehyde and osmium tetroxide, stained with 2% tannic acid, and dehydrated with gradient ethanol. Excessive isoamyl acetate, the samples were dried by HCP-2 critical point dryer and E- 102 vacuum ion sputtering instrument produced by Hitachi, and then observed and photographed by S-4800 scanning electron microscope.

Bear fruit

2. 1 Histomorphological observation

Due to individual differences, guinea pigs have different sensitivities to drugs. The ABR threshold of 13 guinea pigs in 25 experimental groups was greater than 95 dB SPL after 1 week. Scanning electron microscopic observation of cochlea of these severely deaf guinea pigs showed that the static cilia of inner and outer hair cells in the first and second circuits were scattered and fused, and even all hair cells were replaced by scars (figure 1). Light microscope observation of cochlear sections of deaf guinea pigs showed that cochlear hair cells were seriously damaged, mainly outer hair cells. The damage of cochlear hair cells in the first and second circuits is more serious than that in the third and fourth circuits, and some guinea pigs are seriously damaged. Inner hair cells are also generally absent (Figure 2).

2.2 Immunofluorescence histological observation

Five guinea pigs (65,438 00 ears) with ABR threshold greater than 95 dB SPL at different time points after administration were stained with immunofluorescence. Under the laser scanning * * * focusing microscope, it was found that compared with the normal control group, the nerve fibers in both ears of guinea pigs were almost normal except 65,438+0 weeks after administration and 65,438+0 weeks after administration. Similar to the damage of hair cells, the nerve fiber damage in the first and second circuits of cochlea is more serious than that in the third and fourth circuits (Figure 3).

3 discussion

The ototoxicity of aminoglycoside antibiotics is closely related to the toxic effect of oxygen free radicals [4], and loop diuretics can cause angiostriosis and damage the function of lymph secretion [2]. When the above two drugs are used together, aminoglycoside antibiotics contact the inner ear hair cell membrane, which increases the permeability of the inner ear hair cells, while loop diuretics enter the cells in high concentration, causing damage to the hair cells [5].

1979 Russell et al [6] first applied KM and uric acid to guinea pigs. Asakuma et al [7] studied the effects of furosemide and uric acid on the DC potential of guinea pig cochlea with and without kanamycin sulfate.

Bobbin et al. [8] studied the release of γ -aminobutyric acid (GABA) and other substances in guinea pig cochlea induced by potassium by high performance liquid chromatography (HPLC). Guinea pig cochlea was perfused with artificial external lymph with normal K+ concentration (5 mmol/L) and high K+ concentration (50 mmol/L), including normal animals and Corti organ damaged by kanamycin and uric acid in advance. Action-aminobutyric acid, 2- aminoethanesulfonic acid, glutamic acid, aspartic acid and glycine were significantly higher than those in normal potassium concentration group. Compared with the normal group, the release of GABA, 2- aminoethanesulfonic acid, glutamic acid, aspartic acid and glycine induced by potassium in Corti organ destruction group was significantly reduced. From the result analysis, it is considered that the release of GABA is consistent with its neurotransmitter in cochlea.

Raphael et al [9] studied the structural and molecular changes of guinea pig cochlea after ototoxic drugs were used. It was found that the actin filaments on the epidermis and static cilia of cochlear hair cells disappeared, and the bridge structure rich in actin appeared in the top area of dying hair cells. The two supporting cells formed scars at the position of protohair cells, and the supporting cells expanded and invaded the Nuel space and the area where the previous hair cells were located. Scar areas can be marked with cytokeratin. It is also found that the final degeneration site is the tip of hair cells, and the degeneration of hair cells occurs simultaneously with scar formation. In this study, we also found that the sensory epithelial area was completely replaced by scar in the severely damaged guinea pig cochlea by scanning electron microscope (figure 1).

From our previous experimental results, it can be seen that the deafness caused by the combination of KM and furosemide is bilateral symmetry, and after 1 week, half of guinea pigs will have severe sensorineural deafness [3]. The pathological study of cochlea of these deaf guinea pigs found that cochlear hair cells were seriously damaged, mainly outer hair cells, and the damage of cochlear hair cells in the first and second circuits was more serious than that in the third and fourth circuits. Cochlear nerve fiber staining showed that the nerve fibers in deaf guinea pigs decreased significantly, which also showed that the first and second circuits decreased more seriously than the third and fourth circuits. To explore the mechanism of hair cell damage caused by KM and furosemide, it may be that the ability of outer hair cells to swallow ototoxic drugs is stronger than that of inner hair cells, and the ability of outer hair cells in basal gyrus and parietal gyrus to ingest ototoxic drugs is different. As for why hair cells in different parts have different drug uptake capacity, it is speculated that it may be related to the distribution and activity of drug transporters on the cell membrane [4].

Dong Minsheng's experiment proved that after 6 days of continuous intramuscular injection of KM, the sensory cells in the inner ear were damaged first, and the degeneration of sertoli cells was obviously later than that of hair cells, and the degeneration of spiral ganglion and nerve fibers was later. Therefore, it was considered that the damage of sensory cells in the inner ear was the fundamental cause of hearing damage caused by KM. Our experimental results are consistent with the damage of sensory cells in the inner ear, but we find that not only the sensory epithelium of the inner ear but also the auditory nerve fibers are damaged after KM and furosemide are used together.

In 2004, Nourski et al. [1] reported that KM and uric acid were used to establish an animal model of acute deafness, and the efficiency of this method was observed in acute guinea pig experiments, and the auditory sensitivity and auditory nerve state were detected. Therefore, acoustic evoked compound action potential (ACAP) and electrical evoked compound action potential (ECAP) were repeatedly detected. The results showed that the amplitude of ACAP decreased to zero in 4 ~ 6 hours after uric acid administration in 4 of 6 guinea pigs, while ACAP continued to react in the other 2 guinea pigs within 65,438 00 hours after uric acid administration. At the same time, the author also recorded ECAP. Unlike ACAP, the amplitude of ECAP is relatively constant in each experiment, and it is proved that there is no change consistent with the time after administration or the effect of ACAP. Based on the results of ACAP and ECAP, the author concludes that KM and uric acid drugs have targeted damage to hair cell function, but have not significantly inhibited auditory nerve reactivity, which seems to contradict some of our experimental results. The reason for this difference is that the observation time after medication is different: Nourski et al.' s observation time is within 10 hour after medication, while our observation is that the auditory nerve fiber may not be damaged within 1 0 hour after medication, but it begins to be damaged at 1 week, and the nerve fiber is obviously damaged after medication for 3 weeks.

refer to

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2 Dong Minsheng, Dong Mingmin, Lou. Research progress of inner ear diseases. Zhengzhou: Henan Medical University Press, 1999: 12 -22.

Zhang Xi Anfen, Shi Yang Ming, Hu Yinyan, et al. Study on hearing function of guinea pig cochlea after combined application of kanamycin and furosemide. Chinese scientific journal of hearing and speech rehabilitation, 2008, 6 (2): 15-20.

4 Ding Dalian, Salvi. Study on ototoxicity of aminoglycoside antibiotics. China Journal of Otology, 2007,5 (2):125-131.

5 Zhang Yamei. Drug deafness. Chinese Journal of Pediatrics, 2000,38 (12): 781-782.

6 Russell, New Jersey, Foxco, Brummett RE. Otototoxic effect of interaction between kanamycin and ethacrylic acid. Cochlear ultrastructure is related to cochlear potential and kanamycin level. Journal of Otolaryngology,1979,88 (5-6): 369-381.

7 shallow s, snow JB. Effects of furosemide and ethacrylic acid on direct current potential in cochlea of guinea pigs treated with normal and kanamycin sulfate. O tolaryngol Head and Neck Surgery,1980,88 (2):188-193.

8-spool RP, Ceasar G, Fallon M. Potassium induces the release of GABA and other substances in guinea pig cochlea. Hear the resolution,1990,46 (1-2): 83-93.

9 Raphael Y, Altschuler Ridge. Scar formation after drug-induced cochlear injury. Hear Res, 199 1, 51(2):173-183.