OBJECTIVETo generate transgenic mice of NKCC1 +/- (heterozygous) and NKCC1 +/+ (wild-type) that have a targeted disruption in the NKCC1 gene in order to investigate the relationship of one copy of NKCC1 gene (NKCC1 +/-) and age-related hearing loss (AHL) and to study the possible pathogenesis of AHL METHODS: Auditory function of NKCC1 +/- mice was detected regularly by auditory brain response (ABR) and endocochlear potential (EP). Morphology of cochlea was observed by scanning electron microscope and content of NKCC1 protein was detected by Western blot.

RESULTSThe mean value for ABR thresholds was elevated in NKCC1 +/- mice more than that of NKCC1 +/+ mice (P < 0.01). A progression of age-related hearing loss was found in NKCC1 +/- mice. Compared with younger NKCC1 +/- mice, the mean value for ABR thresholds in aged NKCC1 +/- mice was significantly increased (P < 0.05). The EP of NKCC1 +/- aged mice was also significantly decreased more than that of the younger NKCC1 +/+ mice (P < 0.05). And content of NKCC1 protein were reduced with the growth of the age. The scanning electron microscope showed a kind of scattered punctiform absence of outer hair cells in elder NKCC1 +/- mice cochlea.

CONCLUSIONSNKCC1 gene maybe takes part in the pathogenesis of AHL. Mice that expressed only one copy of NKCC1 could lead to AHL. AHL may be correlative with the amounts of NKCC1 protein and its function and also with the loss of outer hair cells perhaps.

Age Factors ; Aging ; genetics ; physiology ; Animals ; Hearing Disorders ; etiology ; genetics ; Heterozygote ; Mice ; Mice, Knockout ; Mice, Transgenic ; Sodium-Potassium-Chloride Symporters ; genetics ; Solute Carrier Family 12, Member 2

Objective: To investigate the expression of cation chloride cotransporter (NKCC1/KCC2) in the neurons from cerebral lesions of children with focal cortical dysplasia (FCD) type Ⅱ, to provide a morphological basis for revealing the possible mechanism of epilepsy. Methods: Eight cases of FCD type Ⅱ diagnosed at Beijing Haidian Hospital, Beijing, China and 12 cases diagnosed at Xuanwu Hospital, Capital Medical University, Beijing, China from February 2017 to December 2019 were included. The expression of NKCC1 and KCC2 in FCD type Ⅱa and FCD type Ⅱb was detected using immunohistochemistry and double immunohistochemical stains. The average optical density of NKCC1 in dysmorphic neurons and normal neurons was also determined using immunohistochemical staining in FCD type Ⅱa (10 cases). Results: The patients were all younger than 14 years of age. Ten cases were classified as FCD type IIa, and 10 cases as FCD type Ⅱb. NKCC1 was expressed in the cytoplasm of normal cerebral cortex neurons and KCC2 expressed on cell membranes. In dysmorphic neurons of FCD type Ⅱa, expression of NKCC1 increased, which was statistically higher than that of normal neurons (P<0.01). Aberrant expression of KCC2 in dysmorphic neurons was also noted in the cytoplasm. In the FCD Ⅱb type, the expression pattern of NKCC1/KCC2 in dysmorphic neurons was the same as that of FCD type Ⅱa. The aberrant expression of NKCC1 in balloon cells was negative or weakly positive on the cell membrane, while the aberrant expression of KCC2 was absent. Conclusions: The expression pattern of NKCC1/KCC2 in dysmorphic neurons and balloon cells is completely different from that of normal neurons. The NKCC1/KCC2 protein-expression changes may affect the transmembrane chloride flow of neurons, modify the effect of inhibitory neurotransmitters γ-aminobutyric acid and increase neuronal excitability. These effects may be related to the occurrence of clinical epileptic symptoms.
Child ; Humans ; Brain/pathology* ; Cations/metabolism* ; Chlorides/metabolism* ; Epilepsy/metabolism* ; Malformations of Cortical Development, Group I/metabolism* ; Solute Carrier Family 12, Member 2/metabolism* ; Symporters/metabolism*

OBJECTIVETo investigate the auditory function and the role of NKCC1 and alpha2 Na, K-ATPase in the potassium recycling of cochlea.

METHODSNKCC1(+/-) / alpha2 Na, K-ATPase(+/-) mice model was established from NKCC1(+/-) and alpha2 Na, K-ATPase(+/-) mice. The auditory function of all strain mice were detected by auditory brainstem response (ABR) and endocochlear potential (EP) to investigate the role of NKCC1 and alpha2 Na, K-ATPase in the potassium recycling of cochlea. Furosemide and ouabain were applied to block the two channels in Castel mice line which can long-time maintain normal auditory function and then their auditory function was detected by ABR to authenticate the mode of potassium recycling in vivo and the relationship between cochlear potassium recycling and NKCC1(+/-) and alpha2 Na, K-ATPase.

RESULTSThe mean value for ABR thresholds in response to stimulus was elevated in NKCC1(+/-) and alpha2 Na, K-ATPase (+/-) mice [(38.49 +/- 12.29) dB and (53.32 +/- 7.62) dB) ] respectively, which was significantly increased compared with that observed in wild type mice [(23.13 +/- 3.78) dB, P < 0.05) ]; The EP value of NKCC1(+/-) [(78 +/- 7) mV] and alpha2 Na, K-ATPase(+/-) mice [(71 +/- 14) mV] was decreased compared with that of NKCC1(+/-) / alpha2 Na, K-ATPase(+/-) mice [( 86 +/- 11) mV]. The auditory function of NKCC1(+/-) / alpha2 Na, K-ATPase(+/-) mice could simulate the model of cochlear potassium recycling well. NKCC1 and Na, K-ATPase were great of importance in the potassium recycling, while the two ion channels were in restrict dynamic equilibrium. Castel mice line after administration with furosemide developed significant ABR threshold shifts (P < 0.05) compared with control group. Castel mice line after administration with ouabain also developed greatly significant ABR threshold shifts (P < 0.05) compared with control group. ABR threshold shifts in mice after administration both furosemide and ouabain was attenuated compared with only administration with furosemide (P < 0.01).

CONCLUSIONSIon channel NKCC1 and alpha2 Na, K-ATPase played important roles in the inner ear potassium recycling. Dysfunction of either of them could influence potassium concentration in the endolymph and lead to hearing loss subsequently. The role of NKCC1 and alpha2 Na, K-ATPase in cochlear potassium recycling was authenticated in vivo. The two ion channels contribute the key role for dynamic equilibrium in cochlear potassium recycling and are of great importance for the metabolism of potassium in the inner ear to maintain the normal auditory function.

Animals ; Auditory Threshold ; Cochlea ; metabolism ; physiology ; Evoked Potentials, Auditory, Brain Stem ; Genotype ; Mice ; Mice, Knockout ; Potassium ; metabolism ; Sodium-Potassium-Chloride Symporters ; metabolism ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Solute Carrier Family 12, Member 2

OBJECTIVETo establish a mice model of aminoglycoside antibiotics (AmAn) induced ototoxicity. Then to investigate the sensitivity of AmAn induced ototoxicity in three mouse strains and effect of kanamycin on the expression of Na-K-2Cl co-transporter-1 (NKCC1) in stria vascularis.

METHODSC57BL/ 6J, CBA/CaJ, NKCC1 +/- mice (each of twenty-four) were randomly divided into four experimental groups A, B, C and D (A kanamycin alone, B kanamycin plus 2, 3-dihydroxybenzoate, C 2, 3-dihydroxybenzoate alone, D control group). Mice were injected with kanamycin or/and 2, 3-dihydroxybenzoate for 14 days. Auditory function was measured by auditory brainstem response (ABR) and morphology of cochlea was observed by succinate dehydrogenase staining. Expression of NKCC1 was detected by immunohistochemistry.

RESULTSMice in group A developed significant ABR threshold shifts (P < 0.01), which were accompanied by out hair cells loss. Mice in group B significantly attenuated ABR threshold shifts with out hair cells loss (P <0.01). The immunostaining of NKCC1 in stria vascularis was attenuated significantly in group A compared with group D (P < 0.01) while the immunostaining in group B was enhanced than which in group A (P < 0.01). CBA/CaJ mice has the highest sensitivity to AmAn in three mouse strains.

CONCLUSIONSAn mouse model of AmAn induced ototoxicity could be established by administration of kanamycin. Kanamycin could inhibit the expression of NKCC1 in stria vascularis. 2, 3-dihydroxybenzoate could attenuate AmAn induced ototoxicity maybe by enhancing the expression of NKCC1. Mice that had the characteristic of presbycusis didn't show additional sensitivity of AmAn induced ototoxicity.

Animals ; Anti-Bacterial Agents ; toxicity ; Blood Vessels ; drug effects ; metabolism ; Cochlea ; blood supply ; drug effects ; metabolism ; Kanamycin ; toxicity ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Parabens ; toxicity ; Sodium-Potassium-Chloride Symporters ; metabolism ; Solute Carrier Family 12, Member 2

OBJECTIVETo explore the relationship of gentamicin-induced cochlear damage with autophagy-related protein LC3, beclin1, Na(+-)K(+-)2Cl(-) cotransporter (NKCC1) mRNA and endothelin-1 (ET-1), and investigate the protective mechanism of PPTA against gentamicin-induced cochlear damage.

METHODSSixty guinea pigs were randomly divided into control group (with saline and artificial perilymph injections), model group (with gentamicin and artificial perilymph injections), concurrent treatment group (with gentamicin and PPTA injections), model control group (with artificial perilymph injection 7 days after gentamicin injection) and delayed treatment group (with PPTA injection 7 days after gentamicin injection). Saline and gentamicin (160 mg/kg) were injected intraperitoneally, and artificial perilymph and PPTA were injected into the otocysts on a daily basis for 7 consecutive days. Hearing impairment of the guinea pigs was analyzed with ABR, and the protein expressions of beclin1 and LC3 in cochlear tissue were tested. The expression of NKCC1 mRNA was detected with RT-PCR, and the expression of ET-1 was detected immunohistochemically.

RESULTSThe ABR thresholds in the model group and model control group were similar (P>0.05) , but significantly higher than those in the other 3 groups (P<0.05); the threshold was significantly lower in concurrent treatment group than in delayed treatment group (P<0.05). Compared with those in the other 4 groups, the expressions of LC3 II, beclin1, and NKCC1 mRNA were significantly increased in the model group (P<0.05); and those in delayed treatment group were significantly lower than those in the model control group (P<0.05). The expressions of ET-1 in the Corti organ, striavascularis and spiral ganglion were significantly higher in the model group but significantly lower in the control group than those in the other 4 groups; ET-1 expression was significantly lower in delayed treatment group than in the model control group.

CONCLUSIONPPTA offers protection against gantamicin-induced cochlear damage in guinea pigs by inhibiting cell autophagy and suppressing of NKCC1 and ET-1 expressions. Early intervention with PPTA produces better therapeutic effect, suggesting that gantamicin causes irreversible injury of the auditory cells.

3,4-Methylenedioxyamphetamine ; analogs & derivatives ; pharmacology ; Animals ; Apoptosis Regulatory Proteins ; metabolism ; Beclin-1 ; Cochlea ; drug effects ; Endothelin-1 ; metabolism ; Gentamicins ; adverse effects ; Guinea Pigs ; Hearing Loss ; chemically induced ; prevention & control ; Microtubule-Associated Proteins ; metabolism ; Solute Carrier Family 12, Member 2 ; metabolism

OBJECTIVETo investigate the age-related expression of KCNQ1 and NKCC1 ion transporters in the stria vascularis in the cochlea of C57BL/6J mice, and to analyze the relationship between the two ion transporters and age-related hearing loss.

METHODSAuditory function of C57BL/6J mice was measured by auditory brainstem response (ABR) at the ages of 4, 8, 14, 24, 40 weeks old respectively. The location of KCNQ1 and NKCC1 ion transporters in the cochlea of C57BL/6J mice was detected by immunohistochemistry staining. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the levels of KCNQ1 and NKCC1 mRNA in the cochlea of C57BL/6J mice at different ages.

RESULTSThe mean values for ABR thresholds in response to click, 4 kHz and 8 kHz sound stimulus of C57BL/6J mice gradually increased with age. The ABR thresholds of the mice of over 14 weeks age were significantly elevated in comparison with lower ages (P < 0.05). In the lateral wall of C57BL/6J mice cochlea, the KCNQ1 protein was mainly expressed at the apical membrane of the strial marginal cells. The localization of NKCC1 protein was mainly present at the basolateral membrane of the stria marginal cells, spiral ligament and the fibrocytes in the inferior portion of spiral limbus. Expression of KCNQ1 and NKCC1 protein in cochlea of C57BL/6J mice showed age-related decreasing. The level of KCNQ1 and NKCC1 mRNA in cochlea of C57BL/6J also showed a age-related decreasing trend. There was a significant reducing of KCNQ1 mRNA level between C57BL/6J mice of 40 and 4 weeks old (P < 0.05). In comparison with the C57BL/6J mice of 4 weeks old, the NKCC1 mRNA levels of 24 and 40 weeks old also showed significant reducing (P < 0.05).

CONCLUSIONSThe mean value for ABR thresholds of C57BL/6J mice gradually increased with age. Expression of KCNQ1 and NKCC1 protein in the stria vascularis of C57BL/6J mice decreases with age. The levels of KCNQ1 and NKCC1 mRNA in cochlea of C57BL/6J showed a age-related reducing trend. Regulating after post-translation may also participate in the adjusting of the age-related decreasing of KCNQ1 and NKCC1 protein in the cochlea of C57BL/6J mice. KCNQ1 and NKCC1 ion transporters may play a critical role in maintaining normal hearing function of inner ear.

Age Factors ; Animals ; Cochlea ; metabolism ; physiology ; Evoked Potentials, Auditory, Brain Stem ; KCNQ1 Potassium Channel ; metabolism ; Mice ; Mice, Inbred C57BL ; Sodium-Potassium-Chloride Symporters ; metabolism ; Solute Carrier Family 12, Member 2

BACKGROUNDAfter establishing a murine model of aminoglycoside antibiotic (AmAn) induced ototoxicity, the sensitivity of AmAn induced ototoxicity in three murine strains and the effect of kanamycin on the expression of Na-K-2Cl cotransporter-1 (NKCC1) in stria vascularis were investigated.

METHODSC57BL/6J, CBA/CaJ, NKCC1(+/-) mice (24 of each strain) were randomly divided into four experimental groups: A: kanamycin alone; B: kanamycin plus 2, 3-dihydroxybenzoate; C: 2, 3-dihydroxybenzoate alone; and D: control group. Mice were injected with kanamycin or/and 2, 3-dihydroxybenzoate twice daily for 14 days. Auditory brainstem response (ABR) was measured and morphology of cochlea delineated with succinate dehydrogenase staining. Expression of NKCC1 in stria vascularis was detected immunohistochemically.

RESULTSAll three strains in groups A and B developed significant ABR threshold shifts (P < 0.01), which were accompanied by outer hair cell loss. NKCC1 expression in stria vascularis was the weakest in group A (A cf D, P < 0.01) and the strongest in groups C and D (P < 0.05). CBA/CaJ mice had the highest sensitivity to AmAn.

CONCLUSIONSAdministration of kanamycin established AmAn induced ototoxicity. Kanamycin inhibited the expression of NKCC1 in stria vascularis. 2, 3-dihydroxybenzoate attenuated AmAn induced ototoxicity-possibly by enhancing the expression of NKCC1. Age related hearing loss did not show additional sensitivity to AmAn induced ototoxicity in murine model.

Animals ; Anti-Bacterial Agents ; toxicity ; Auditory Threshold ; drug effects ; Hair Cells, Vestibular ; drug effects ; Kanamycin ; toxicity ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Sodium-Potassium-Chloride Symporters ; analysis ; drug effects ; Solute Carrier Family 12, Member 2 ; Stria Vascularis ; chemistry ; drug effects