Effects And Mechanism Of Ketamine On The Electrophysiological Properties In Cell Level Of Isolated Outer Hair Cells (OHCs) From The Cochlears Of SD Rats In Vitro

General anesthetics are widely used in clinic,but could significant impact on a variety of physiological functions of patients in perioperative period, especially feeling function.Auditory sense as a important way for human to feeling the information outside, tend to be disappear lastly in the process of general anesthesia, and recover firstly in anesthesia awakening period. Thus auditory function as a important indicator often used to estimate the depth of anesthesia in clinical practice.To investigate the influence of anesthetics on auditory function has a great clinical significance,also got more and more attention.In clinical anesthesia, some patients appeared kind of hearing loss, some of them with clinical symptoms can be found, but the overwhelming majority of them is subclinical, can be found only through hearing specialized examination, often ignored by anesthesiologist.In the process of anesthesia,many factors can affect hearing, such as anesthesia method, extracorporeal circulation, ototoxic drugs, hemodynamic changes, middle ear pressure changes, etc.Current research suggests, nitric oxide can cause hearing loss, ketamine can cause hallucination, tinnitus.The effect of general anesthetics on the auditory system can be respectively affect the auditory nerve centre and the peripheral auditory function.At present a large number of animal experiments and clinical studies were focused on central level, about the study of effects of general anesthetics on the peripheral auditory system are indirect detection cochlear ear acoustic emission to infer the role of Peripheral auditory receptors hair cells function.there is no use to separate cells by patch clamp method to study the research of the general anesthetics on hair cell function.There is no study using patch clamp technique to investigate the effect of general anesthetics on hair cell function so far.It had been confirmed that salicylic acid, aminoglycoside antibiotics, furosemide, etc drugs could damage OHCs by patch clamp technique.Therefore, this research intends to detect the influence of ketamine on OHCs (cochlear amplifier) electrophysiological characteristics and to explore its mechanism using patch clamp technology.Objective To study the effects and mechanism of ketamine on the electrophysiological properties in cell level of isolated outer hair cells (OHCs) from the cochlears of Sprague Dawley (SD) rats in vitro. Provide theoretical basis and new ideas for clinical prevention and treatment of perioperative temporary or permanent hearing loss.Methods This is self controlled study in order to avoid individual differences. Acute isolated outer hair cells of SD rats were performed. In this study self-matching compared was used. The datas of whole cell currents were collected using the whole cell patch clamp recording techniques and analyzed.40 healthy adult SD rats,18～21 days age,40～70 g, either sex, were rapidly decapitated. Acute mechanically separated and digested OHCs of cochlears were performed. Vigorous OHCs were randomly chosen and self-matching compared in this study. Whole-cell currents and reversal potential were recorded using whole-cell patch clamp techniques. Experiment includes five parts:(1) Observe the influence of the drug buffing methods on whole-cell current of OHCs:Holding OHCs at -70mV,whole-cell currents stimulated by commanded voltages of 5 OHCs were compared between group C (no buffing drug) and group L (buffing drug L-15).(2) Fit ketamine concentration curve and select the experiment concentration of ketamine:Holding cells at 3mV,the ketamine-sensitive currents of 5 OHCs induced by 1 μM/L,10 μM/L,100 μM/L,1000 μM/L,5000 μM/L ketamine,then fit ketamine concentration curve.(3) Observe the effect of 100 μM/L ketamine on Ⅰ-Ⅴ curve and resting membrane potential of OHCs:Holding OHCs at-70mV, whole-cell currents of 10 OHCs stimulated by commanded voltages were recorded with drug buffing nothing (group N),100 μM/L ketamine after 1 min (group K), nothing after 1 min (group N’) within extracellular fluid L-15.After observed phenomenon, current clamp 10 cells at 0 pA, comparing two groups of cells resting membrane potential.(4) Observe the effect of 100 μM/L ketamine on IACh of OHCs:Holding cells at 3mV,steady IACh of 10 OHCs were recorded with drug buffing 100 μM/L ACh 15s when extracellular perfusing L-15 (group K1), after 1 min perfusing 100 μM/L ketamine (group K2) and after 1 min eluting with L-15 (group K3).(5) Observe the change of ketamine-sensitive currents of OHCs after blocking nAChR using strychnine:Holding OHCs at 3mV,steady ketamine-sensitive outward currents of 10 OHCs were compared with drug buffing 100 μM/L ketamine 15 s when extracellular perfusing L-15 (group S1), after 1 min perfusing 0.1 μM/L strychine (group S2) and after 1 min eluting with L-15 (group S3).Whole-cell currents filtered at 5 kHz. The signal was amplified using an amplifier. Photodiode signals were low-pass filtered (1200 Hz) before delivered to a D/A converter. The signals were acquired by software pClamp 10.3 running on an compatible computer and a A/D converter. Experiments were performed at room temperature (23～25℃).Results Cells were selected for experiment only if its length was approximately constant throughout its diameter and if it showed no signs of damage, such as blebbing, swelling or dislocation of the nucleus. During the experiment cells were excluded if visible appearance changes occurred and signs of damage.(1) The typical Ⅰ-Ⅴ curve of OHC in the L-15 is shown as total currents are inward in cell hyperpolarization direction (left of reverse voltage) and outward in depolarization direction (right of reverse voltage). Inward currents are smaller, outward currents get to bigger and bigger in stimulating voltage depolarization to about more than-50 mV with apparent voltage dependence and outwardly rectifying. Comparing the 1-V curves of four OHCs between group C and L, we found that total current under each membrane voltage had no statistical difference, suggesting that drug delivery mode (drug flow) did’t influence the phenomenon in follow-up study.(2) The effect of different concentration of ketamine on a typical OHC:Cell total current amplitude change of 0 pA with buffering 1 μM/L ketamine,17 pA with 10 μM/L,80 pA with 100 μM/L,153 pA with 1000 μM/L and 184 pA with 5000 μM/L. The decrease in the total outward current amplitude was increases with ketamine concentration increases.The phenomenons of 5 OHCs were consistent.Drawing the ketamine concentration curve taken from average ketamine sensitivity current amplitude from five OHCs fit by Hill equation (IC50=117.3 μM/L). Cells was the most sensitive under 100 μM/L ketamine, so 100 μM/L ketamine was chosen for later experiment.(3) Drawing the Ⅰ-Ⅴ curves of group N and K,we found that compared to group N, the total currents amplitude in group K were reduced voltage-dependent in membrane potential depolarized more than-36 mV, but the proportion of current reduce under various voltages is certain (15.5%～17.5%). The average reversal potentials of two groups had no statistical differences (P> 0.05). The reversal potential of ketamine-sensitive current was 55.2±6.1 mV, similar to potassium balance potential, therefore, we speculated that the effect of ketamine on OHCs by influencing potassium currents.The resting potentials of 10 OHCs from 2 groups had no statistical differences.(4) 100 μM/L ketamine had no effect on IACh induced by 100 μM/L acetylcholine (ACh) of 10 OHCs at holding potential 3mV (P> 0.05).(5) Perfusing a9 nAChR blocker strychnine (0.1 μM/L), the amplitude of ketamine-sensitive currents didin’t change at holding potential was 3mV (P> 0.05).Conclusion 1. Ketamine can restrain OHCs membrane currents with concentration dependent.2.100 μM/L ketamine could voltage-dependent reduced the total currents amplitude in membrane potential depolarized more than-36 mV, but the proportion of current reduce under various voltages is certain. The average reversal potentials and resting potentials of two groups had no statistical differences. The result suggesting that ketamine didn’t influence electromobility of OHCs by the way of changing membrane potential.3. Combination of activation potential of ion channels and currents in OHCs, we presumed that the effect of ketamine on OHCs may be through inhibiting calcium dependent potassium current.4. One research had found that 100 μM/L ketamine could decrease the intracellular calcium concentration of OHCs, thus we speculated that ketamine decreases OHC electromobility by inhibiting the intracellular calcium concentration, restraining membrane proteins cortical cytoskeleton and finally increasing the global axial stiffness of the cell.