Mycoplasma species. We investigated the antibacterial activity of doxycycline against the Mycoplasma hyopneumoniae strain ATCC25934. The minimum inhibitory concentration (MIC) of doxycycline against M. hyopneumoniae determined by a microdilution method was 0.125 μg/ml. Static time–kill curves with constant drug concentrations (0–64 MIC) showed that a bacteriostatic effect occurred if the doxycycline concentration reached 4 MIC. Doxycycline produced a maximum antimycoplasmal effect (reduction of 2.76 log10CFU/ml) at 64 MIC within 48 h. The effect of doxycycline against M. hyopneumoniae was analyzed by a sigmoid Emax model, and there was high correlation between the kill rate and doxycycline concentration (R2 = 0.986). A one-compartment open model with first-order absorption was adopted and was used to simulate doxycycline pharmacokinetics in porcine plasma. The dynamic time–concentration curve showed that the area under the curve at 24 h (AUC24 h) and Cmax (peak concentration) after each drug administration was 1.78–48.4 μg h/ml and 0.16–3.41 μg/ml, respectively. The reduction of M. hyopneumoniae (log10CFU/ml) for 1, 2.5, 5, 7.5, 10, 15, 20, and 30 mg/kg body weight was 0.16, 1.29, 1.75, 2.94, 3.35, 3.91, 4.35, and 5.77, respectively, during the entire experiment, respectively. When the dose was >10 mg/kg body weight, continuous administration for 3 days could achieve a bactericidal effect. The correlation coefficient of AUC24 h/MIC, Cmax/MIC, and %T > MIC (the cumulative percentage of time over a 24-h period that the drug concentration exceeds the MIC) with antibacterial effect was 0.917, 0.923, and 0.823, respectively. Doxycycline showed concentration-dependent activity, and the value of AUC24 h/MIC and Cmax/MIC required to produce a drop of 1 log10CFU/ml was 164 h and 9.89, respectively.Doxycycline is a broad-spectrum antibacterial drug. It is used widely to treat diseases caused by  Mycoplasma hyopneumoniae is the primary etiologic agent of enzootic pneumonia and is widespread in pig populations  interactions of antibacterial agents against Mycoplasma species, including doxycycline. However, in recent years, development of an in vitro PK/PD model has produced a robust method for correlation research  in vitro. The dilution model they used was simulated by increasing the volume, which cannot mimic the continuous process of drug elimination. Mycoplasma gallisepticum using a simple in vitro dynamic model that could eliminate the agent through an outlet, and the initial antimicrobial concentrations corresponded to various multiples of the minimum inhibitory concentration (MIC).Studies of treatment of pigs infected on a farm have indicated that doxycycline has significant therapeutic activity against mycoplasmal pneumonia . The nutresearch . MitchelM. hyopneumoniae based on PK/PD interactions. The model was based on the studies mentioned above with some necessary modifications: (i) we added an absorption chamber to mimic absorption in animals, and (ii) the design of the dose regimen was in accordance with clinical practice. We wanted to provide a reference for dose-regimen optimization which could achieve a cure for Mycoplasma pneumoniae infection and minimize the opportunity for antimicrobial resistance.We investigated the effect of doxycycline against M. hyopneumoniae strain ATCC25934 was determined; (ii) the in vitro time–kill curves for doxycycline against M. hyopneumoniae in a defined artificial medium were established and the relationship between the kill rate and drug concentrations were fitted to the Emax model (see below); (iii) the relationship between PK/PD indices and the effect of doxycycline against M. hyopneumoniae were investigated in an in vitro dynamic PK/PD model; and (iv) a scientific dosing guidance of doxycycline against M. hyopneumoniae was provided based on PK/PD modeling.We pursued four objectives: (i) the MIC of doxycycline against M. hyopneumoniae (ATCC25934) was provided by the Chinese Veterinary Microorganism Culture Collection Center . A doxycycline hyclate standard (>99%) was obtained from the China Institute of Veterinary Drug Control . The artificial medium base of M. hyopneumoniae was purchased from Qingdao Hope Biological Technology . Sterile pig serum was supplied by Guangzhou Ruite Biological Technology . Cysteine and the reduced form of nicotinamide adenine dinucleotide were supplied by Guangzhou Prob Information Technology. The “raw” form of doxycycline (87.4%) was provided by Guangdong Dahuanong Animal Health Products .The standard strain of M. hyopneumoniae strain ATCC25934, as described by 6 CFU/ml. The culture medium of M. hyopneumoniae contained artificial medium base, 20% pig serum, trace cysteine, and nicotinamide adenine dinucleotide. A series of concentrations of doxycycline was prepared by twofold dilution  with media. Each of the first to the ninth column was added 0.1 ml of media with different concentrations of doxycycline and 0.1 ml of M. hyopneumoniae inoculum. A growth control , an end-point control (blank medium at pH 6.8) and a sterility control (sterility medium) were included. After sealing with a gas-permeable film, plates were cultured at 37°C in an atmosphere of 5% CO2. The MIC was determined as the minimum concentration of doxycycline that resulted in no change in color. All experiments were carried out in triplicate.A microdilution method was used to determine the MIC of doxycycline against M. hyopneumoniae at static drug concentrations. The doxycycline concentrations used were 0, 0.5, 1, 2, 4, 6, 8, 16, 32, and 64 MIC. Then, 0.4 ml of the M. hyopneumoniae suspension in the exponential phase, 0.1 ml of drug solution, and 3.5 ml of culture medium were added to a 7-ml penicillin bottle. The initial inoculum size was ∼106 CFU/ml. A growth control  and sterility control (4 ml of blank medium) were included. Cultures were incubated at 37°C in an atmosphere of 5% CO2 for 48 h, and 100 μl of each culture was collected at 0, 1, 3, 6, 9, 12, 24, 36, and 48 h for M. hyopneumoniae counting. Each sample was serially diluted 10-fold, then 10-µl dilutions were transferred to agar plates. The latter were incubated for 7 days at 37°C in a humidified incubator in an atmosphere of 5% CO2. Then, the number of M. hyopneumoniae colonies was recorded using an inverted microscope . The limit of detection was 100 CFU/ml. Time–kill studies were repeated at least thrice on different days.We determined the effect of doxycycline against M. hyopneumoniae were presented by plotting log10CFU/ml against time (h) at different concentrations. First, the kill rates of 0–24 h, 0–36 h, and 0–48 h time periods were calculated. The kill rates of 3–24 h, 3–36 h, and 3–48 h were also calculated because that a recovery growth was observed after transferring to the new culture vessel. Besides, the kill rates were also calculated from 12 and 48 h because there was no significant reduction in M. hyopneumoniae counts within 12 h. Thus, eventually, the mean kill rate for seven time intervals  was calculated by linear regression. The mean kill rate for these seven time intervals was selected to fit the drug concentration using the Emax model employing WinNonlin 6.1 . The Emax model can be presented as follows:The kill rate can reflect the effect of antibacterial drugs against a pathogen. The greater the growth rate, the stronger the antibacterial effect of the drug. The time–kill curves of doxycycline against E is the kill rate, Emax is the maximum value of kill rate in a certain time interval, Ce is the doxycycline concentration, N is the Hill coefficient (which describes the steepness of the kill rate–concentration curve), EC50 is the concentration at which 50% of the maximum kill rate is reached, and R2 is the correlation coefficient of the relationship between experimental values and predicted values.where in vitro dynamic model previously established in our laboratory  were designed in the in vitro dynamic model once daily for 3 days. Sampling was done 0 h before administration as well as 1, 2, 4, 6, 12, 24, 30, 36, 48, 54, 60, 72, 84, and 96 h after administration for determination of the drug concentration). in addition, 0.1 ml of inoculum in the dialysis tube was sampled 0 h before administration as well as 6, 12, 24, 30, 36, 48, 54, 60, 72, 84, and 96 h after administration for bacterial counting. All experiments were carried out in triplicate.Ten milliliters of inoculum of turbidity 106 CFU/ml, and 10 μl of dilution was dropped on the surface of drug plates that contained 1 MIC concentration. After 7 days of culture, the colonies recovered on the plate were inoculated into the liquid medium for further culture. After continuous passaging for five times, the MIC of the strain was determined by a microdilution method. If the MIC value of the strain remained high, it indicates that the strain reduced sensitivity.The samples were collected at 24, 48, 72, and 96 h from a dialysis tube, which were cultured to the logarithmic growth phase. Then, the exponential-phase culture was diluted to the inoculum size of 1024 h) was calculated using the trapezoidal method, and the Cmax after each drug administration was observed directly from the curve. The parameter of %T > MIC were calculated according to a noncompartmental analysis by WinNonlin. Then, data were analyzed using the sigmoid Emax model as follows:PK/PD parameters were derived from time–concentration assays, and the antimycoplasmal effect was obtained from dynamic time–kill curves. The area under the concentration–time curve at 24 h  in periods of 0–24 h after each administration; E0 is the maximum value of the antimycoplasmal effect; Ce is the value of PK/PD indices ; EC50 is the PK/PD index that produces a 50% reduction of the maximum antimycoplasmal effect; and N is the Hill coefficient.where M. hyopneumoniae ATCC25934 as determined by the microdilution method was 0.125 μg/ml.The MIC of doxycycline against 10CFU/ml within 48 h. When the drug concentration was <2 MIC, mild inhibition of M. hyopneumoniae proliferation compared with that in the growth control group was observed, and M. hyopneumoniae showed a slow increase relative to the initial inoculum size. When the drug concentration was >4 MIC, doxycycline showed bacteriostatic activity against M. hyopneumoniae within 48 h but did not show a bactericidal effect at all doses. There was no obvious change in the number of M. hyopneumoniae colonies within 12 h, except for a decrease at <64 MIC, which had a reduction of 0.39 log10CFU/ml. The static time–kill curve is shown in R2 estimated from the different time intervals ranged from 0.970 to 0.986. The maximum R2 (0.986) arose during the period 12–48 h, and the maximum kill rate was 0.11/h. The profile of the kill rate demonstrated that, if the concentration was ≤16 MIC, the kill rate increased with increasing concentration. The relationship between concentration and kill rate was fitted by the Emax model, and the obtained parameters of Emax, EC50, and the Hill coefficient are presented in The relationship between the kill rate and concentration is presented in in vitro dynamic model for doxycycline is displayed in 24 h and Cmax after each drug administration was 1.78–48.4 μg h/ml and 0.16–3.41 μg/ml, respectively. According to the recommended dose of 10 mg/kg body weight, the peak concentration could reach 1.49 μg/ml in a single administration, which is well above the MIC (0.125 μg/ml) of doxycycline against M. hyopneumoniae. Data were analyzed according to a one-compartmental model by WinNonlin and are shown in in vitro dynamic model fitted a one-compartment open model with first-order absorption (R2 = 0.994).The concentration–time curve of the M. hyopneumoniae at eight simulated dosing regimens. The reduction in bacterium (log10CFU/ml) for several dosing regimens  was 0.16, 1.29, 1.75, 2.94, 3.35, 3.91, 4.35, and 5.77 during the entire experiment, respectively. Within the interaction at 24 h, there was no marked decrease in the maximum reduction (0.31 log10CFU/ml). When the dose was <7.5 mg/kg body weight, doxycycline could kill M. hyopneumoniae (maximum reduction = 2.94 log10CFU/ml). When the dose was >10 mg/kg body weight, continuous administration for 3 days could achieve a bactericidal effect. At 30 mg/kg body weight, the number of M. hyopneumoniae could fall below the limit of detection (100 CFU/ml) during the entire experiment. Hence, doxycycline had a significant effect on M. hyopneumoniae, and escalating doses of doxycycline resulted in marked killing of M. hyopneumoniae. There have no drug-resistant strains been obtained under various dosing regimens.Emax relationships of three PK/PD parameters  versus the antimycoplasmal effect are shown in R2 of AUC24 h/MIC, Cmax/MIC, and %T > MIC with an antimycoplasmal effect was 0.917, 0.923, and 0.823, respectively. These results suggested that the effect of doxycycline against M. hyopneumoniae was concentration dependent and that efficacy was driven by AUC24 h/MIC and Cmax/MIC. The relationship between antibacterial efficacy and PK/PD parameters was assessed by a sigmoid Emax model, and the obtained parameters of E0, Emax, and EC50, and Hill coefficient are presented in 24 h/MIC and Cmax/MIC for producing a drop in log10CFU/ml of 1 was 164 h and 9.89, respectively.The 24h/MIC, the area under the curve at 24 h by MIC; Cmax/MIC, peak concentration by MIC; Emax, the change of control sample (log10CFU/ml) in periods of 0–24 h after each administration; E0, the maximum value of the antimycoplasmal effect; EC50, the PK/PD index that produces a 50% reduction of the maximum antimycoplasmal effect.AUCIn vitro dynamic models provide conditions for the studying of PK/PD of mycoplasma. M. hyopneumoniae is an important infectious pathogen in pigs and causes major economic losses to the pig industry. However, relevant studies have been hindered by three main difficulties. First, the characteristics of Mycoplasma species  mean that the nutrients and environment for in vitro culture must be controlled strictly  with other Mycoplasma species  avoided loss of M. hyopneumoniae. Samples could be collected continuously to monitor the growth and death of M. hyopneumoniae.This PK/PD model reflects the relationship between the drug, bacteria, and quantification of the activity (as well as the likely efficacy) of antimicrobial agents against target pathogens . The dynM. hyopneumoniae colonies on agar plates to investigate the effect of doxycycline against M. hyopneumoniae. The in vitro dynamic time–kill curve could characterize the activity of doxycycline against M. hyopneumoniae preliminarily. Where appropriate, data were fitted to the sigmoid Emax model to obtain specific PK/PD parameters, and the PK/PD index obtained was used to calculate the parameter value corresponding to a certain antibacterial effect. We could investigate the PK/PD interactions of doxycycline against M. hyopneumoniae to determine the concentration and/or time dependence of such activity and provide guidance for designing dose regimens for the clinical treatment of M. hyopneumoniae infection.The PK/PD model used in the study is functionally superior to other models. Compared with the older dynamic model using only the central effect chamber, this model adds an absorption chamber to simulate drug absorption. Bacteria were cultured in dialysis tubes that allowed exchange of drug and nutrients . We counM. hyopneumoniae determined using a microdilution method was 0.125 μg/ml. To be consistent with the in vitro experiment of time–kill curves, the final inoculum of M. hyopneumoniae in each well was 106 CFU/ml in our study. The recommended turbidity of MIC testing against veterinary Mycoplasma species is 103–105 CFU/ml  and MIC90  was 0.078 and 0.312 μg/ml, respectively. Moreover, most studies from Europe have demonstrated similar values of MIC50 and MIC90 for oxytetracycline  reduction was 164 h and 9.89, respectively. These data provided a scientific dosing guidance of doxycycline against M. hyopneumoniae.The study has shown that doxycycline has a significant effect on M. hyopneumoniae in an in vitro dynamic model using drug-containing agar plates. No drug-resistant strains were obtained under various dosing regimens during short-term experimental studies. The result indicated that M. hyopneumoniae is not easy to get resistant for doxycycline.There have no drug-resistant strains been obtained in this study. We tried to screen for drug-resistant strains of M. gallisepticum  need longer to elicit their effects.Studies have shown that doxycycline does not have activity against MmmSC if the bacteria are exposed to the drug for 12 and 24 h at a constant concentration . Those fsepticum . In thisic agent . Third, Cmax/MIC parameter had the highest correlation with the antibacterial effects (R2 = 0.923). Therefore, we could infer that the antimicrobial effect of doxycycline against M. hyopneumoniae was concentration dependent. In this study, WinNonlin was employed to fit the drug concentration and kill rate . The results suggest that higher doxycycline concentration result in faster kill rate until the doxycycline concentration was >16 MIC. In traditional PK/PD studies, bacteria are exposed to a constant drug concentration, and efficacy evaluation is based on time–kill curves  and Cmax/MIC (R2 = 0.923). Doxycycline seemed to be driven by the AUC.According to the bactericidal properties of drug on the bacteria, drugs can be divided into time- and concentration-dependent drugs. When the antibiotic concentration is increased, concentration-dependent drugs lead to faster killing, whereas the kill rate of time-dependent drugs might be remained constant . In thisl curves . This meStaphyloccus aureus, Steptococcus pneumonia) and Gram-negative pathogens  suggests that doxycycline kills bacteria by time-dependent kinetics at low concentrations but by concentration-dependent kinetics at high concentrations .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We present a novel method for determination of βdark using local electroretinography (LERG) from isolated mouse retinas. The method is based on the ability of PDE inhibitors to decrease βdark, which can be counterbalanced by increasing PDE activity with light. This procedure clamps cytoplasmic cGMP to its dark value. βdark can be calculated based on the amount of light needed for the “cGMP clamp” and information extracted from the registered rod photoresponses. Here we apply this method to determine βdark values for the first time in the mammalian rods and obtain the following estimates for different mouse models: 3.9 s−1 for wild type, 4.5 s−1 for guanylate cyclase activating proteins (GCAPs) knockout, and 4.4 s−1 for GCAPs and recoverin double knockout mice. Our results suggest that depletion of GCAPs or recoverin do not affect βdark.Light regulates cGMP concentration in the photoreceptor cytoplasm by activating phosphodiesterase (PDE) molecules through a G-protein signalling cascade. Spontaneous PDE activity is present in rod outer segments even in darkness. This basal PDE activity (β A rapid drop in the cytoplasmic cGMP concentration leads to the closure of the cyclic nucleotide gated (CNG) channels in the outer segment plasma membrane, hyperpolarization of the cell membrane, change in the release rate of glutamate in the rod terminal and transmission of the light-generated signal to the inner retina (see e.g.3). Thermal energy causes spontaneous activations of phototransduction molecules, which leads to fluctuations in the cytoplasmic level of cGMP. These fluctuations make up the main part the dark noise of photoreceptors4.Photoreceptor cells convert light information to sensory signals in a process called phototransduction. When a photon is absorbed in a rhodopsin molecule in the rod outer segment disk membrane, the rhodopsin activates G-proteins, transducins, and the activated transducins bind to phosphodiesterase-6 molecules (PDE) forming enzyme complexes, which hydrolyse cyclic guanosine monophosphate (cGMP) at nearly a diffusion limited rate4. The amount of active PDE in darkness determines the rate constant for spontaneous cGMP hydrolysis, i.e. the basal PDE activity (βdark), which sets the steady state level and the turnover rate of cGMP. Hence, it is one of the main factors in setting the kinetics of photoresponse deactivation and spatial propagation of cGMP concentration drop during photoresponses5. The basal PDE activity has been determined earlier for amphibian rod photoreceptors by abruptly blocking the activity of either PDE or guanylate cyclase10. In the method, single photoreceptor outer segment is exposed to rapid solution changes while recording photoreceptor circulating dark current. However, this has turned out to be challenging with the fragile mammalian photoreceptors, and until now, no one has determined the βdark of wild type mammalian photoreceptors. Gross et al. (2012) demonstrated that when the calcium mediated feedback to guanylate cyclase is abolished by knocking out the guanylate cyclase activating proteins (GCAPs) and the lifetime of activated PDE is decreased by overexpressing RGS9, the basal PDE activity becomes the dominant factor determining the light response deactivation kinetics5. In these circumstances, the late recovery of a single-photon response allows the determination of βdark. However, it is not known whether these genetic manipulations, which affect cGMP homeostasis and PDE deactivation kinetics, influence the basal PDE activity and, thus, the βdark value is not directly generalizable to wild type (WT) mice.The dark noise consists mainly of three components: discrete spontaneous activations of rhodopsin, high frequency noise from fluctuations in the CNG channel conductance, and continuous noise from thermal activations of PDEdark for WT mice. The method is based on the ability of PDE inhibitors to decrease both the basal and light-induced PDE activity. A decrease in the basal PDE activity can be compensated for by increasing the light-induced PDE activity in a controllable way14. As a result, the cGMP concentration in the outer segment, CNG channel current, and the photoreceptor signalling stay at constant level. We assume that the amount of light-induced PDE activity needed to keep the photoreceptor signal constant matches with the decrease in βdark. An essential feature in this compensation is that besides the cytoplasmic cGMP also intracellular Ca2+ levels stay clamped in their dark concentrations to avoid modulation of phototransduction.In this study, we introduce and test a novel approach to determine rod β−/− and GCAPs−/− recoverin−/− double knock out mice (DKO). The latter phenotype enables simplified modelling of photoresponses, because the predominant calcium feedbacks to phototransduction are absent15. The βdark value determined with our cGMP clamp method was in line with the βdark value obtained by modelling of GCAPs−/− and DKO photoresponses, and with the value determined for GCAPs−/− in5. The utilization of the new method is not limited to mice but it is applicable in quantitative determination of steady state phosphodiesterase activity regardless of the species or the genetic background of the model animal.In addition to WT mice, the performance of the method was investigated with GCAPs16 and in mice17 in light-independent manner.The turnover rate and the concentration of intracellular cGMP in rod photoreceptor cells are regulated by the rates of hydrolysis of cGMP by PDE and synthesis of cGMP by guanylate cyclase. In the cGMP clamp procedure, a PDE inhibitor is introduced to the retina while monitoring changes in the extracellular voltage with local ERG recording across the outer segment layer (LERG-OS) or across the whole photoreceptors (LERG-PR). In principle, similar recordings could be conducted with transretinal ERG (TERG). However, our preliminary experiments with simultaneous TERG and LERG recordings revealed that IBMX generated an additional sustained component originating in the inner retina in spite of pharmacological blocking of synaptic transmission from rods to bipolar cells. This light-independent change in the TERG signal baseline prevented us from using TERG in cGMP clamp. A possible source for this component is the ON-bipolar cells, because IBMX has been demonstrated to potentiate the ON-bipolar cells in salamandersdark) can be calculated based on the light intensity needed to keep the signal stationary when the phototransduction parameters in equation  activated PDE and leads to an increase in the level of cGMP. However, the decrease in the basal PDE activity can be compensated for by increasing the amount of light-activated PDE with an accurately controlled closed loop background light feedback, thereby keeping the cGMP level clamped in the dark-adapted value , the guanylate cyclase activating protein knockout mice (GCAPs−/−), and the GCAPs and recoverin double knockout mice (DKO).Most of the parameter values needed for βet al.19 introduced a method for determining the dominant time constant, τD, of saturated flash response deactivation, which is demonstrated in Fig. 20. The average τD estimates obtained with the Pepperberg analysis were 191 ± 13 ms (n = 10) for WT, 222 ± 7 ms (n = 6) for GCAPs−/−, and 200 ± 13 ms  for DKO mice.Pepperberg 21, the response onset is assumed to be defined only by the activation phase of the phototransduction and it can be fitted to early onset of the responses until the response termination starts to affect the response shape. In mouse rods, the shortness of the rhodopsin lifetime is the main factor limiting the valid time window for Lamb and Pugh activation model fitting because coincidentally with the deactivation of rhodopsin, the steepness of the photoresponse onset starts to decline. Deactivation of rhodopsin proceeds through a sequence of several phosphorylation steps by rhodopsin kinase, each of which increases the probability for an arrestin molecule to bind and completely deactivate the activated rhodopsin25. Determination of the average lifetime of active rhodopsin (τR) has turned out to be challenging and currently there are no means for its direct determination in mammalian photoreceptors. Nevertheless, τR can be estimated by modelling rod photoresponses. With the simplifying assumption that rhodopsin deactivation follows first order reaction kinetics on average, the mean lifetime for rhodopsin in WT mouse rods is estimated to lie close to 40 ms26. This proposes that the rate of transducin activation will drop to half in less than 30 ms from a brief stimulus, leaving only a very narrow time window to determine the “true” amplification constant of phototransduction before rhodopsin deactivation substantially starts to shape the responses.In Lamb and Pugh activation modelα ≈ 0, and (2) when the following holds: βlight(t)cGMP(t) ≫ βdark(cGMPdark − cGMP(t)) (see Methods for derivation). The first condition holds always with GCAPs−/− and DKO mice, but for WT mice it is true only for the early activation phase of the response. This is demonstrated in Fig. −/− (n = 6) retinas recorded with LERG-OS. The activation phases of the responses to similar flash strengths start to diverge clearly only after 70–100 ms from the response onset as shown earlier for dim flash responses28. So, we assume in our analysis that condition 1 holds at times <70 ms after the flash.To extend the time window for the determination of the amplification constant, we used a model that takes into account the activation reactions as well as deactivation of activated rhodopsin and PDE but disregards the hydrolysis of cGMP by basal PDE activity and synthesis of cGMP by guanylate cyclase (equation ). Equatiβlight(t) and βdark. We set the criterion for the validity of condition (2) to βlight(t)cGMP(t) > 10⋅βdark(cGMPdark − cGMP(t)). We then calculated the maximal time from the flash stimulus that still meets the criterion. To analyse the condition, we calculated βlight(t) with different flash strengths with equations (βlight(t) were chosen to be realistic but at the same time to give rather too narrow than wide range for the validity of equation  was calculated numerically from equation  is less than 10% during the first 34 ms from the beginning of the flash response. The validity time set by the second criterion, 34 ms, is shorter than the one set by the first criterion, 70 ms, and thus, the fitting of the phototransduction model was carried out using only the first 34 ms of the responses from the beginning of the flash stimulus. If our final βdark estimate was higher than 6 s−1, this analysis should be repeated with a tighter criterion for the validity of equation (The fulfilment of the condition (2) depends on both the light-induced and basal PDE activity levels, quations  and 14)βlight(t)equation . The parequation  by deterequation . 200 ms equation  and cGMPequation .Table 1P−/−, and DKO mice, respectively, from all registrations recorded with LERG-OS. Since in the fitting, amplification constant A and rhodopsin average lifetime τR cannot be determined fully independently from each other, we fitted the model with all possible combinations of A and τR. The lifetime of PDE was locked to the values determined as described earlier. Figure −/− and DKO population averaged responses recorded with LERG-OS. Figure −/−, and 28 ms for DKO mice. From now on, the rhodopsin lifetimes were fixed to these estimated values and the amplification constants were determined separately for each retina used in the cGMP clamp experiments. The average amplification constants were 15.8 ± 1.8 s−2 for WT (n = 10), 14.0 ± 1.6 s−2 for GCAPs−/− (n = 6), and 20.6 ± 2.6 s−2 for DKO mice . These values are close to the amplification constant values determined earlier for mouse rods with suction electrode recordings in resembling nutrition medium (range from 8.3 to 23 s−2)31.To maximize the signal-to-noise ratio in our fitting of the phototransduction model to the early phase of light responses, we calculated population averaged flash response families for WT, GCAPsKI,light) was determined based on the decrease in phototransduction amplification caused by the PDE inhibitor as described in32. The inhibition constant was determined separately for each retina and the average KI,light for IBMX was 16.3 ± 1.0 µM for WT (n = 10), 13.2 ± 1.2 µM for GCAPs−/− (n = 6), and 13.8 ± 1.7 µM for DKO  mice. Figure KI,light determination from averaged DKO data.The inhibition constant of IBMX towards light-activated PDE For IBMX, the inhibition constant towards light-activated and spontaneously activated PDE have been found to be very similar in literaturesee Fig.  with theequation  in32)1\dKI,dark determination was done by extrapolating the increase in the relative maximal LERG-OS signal to zero inhibitor concentration. Because the increase in the outer segment current  is modulated by intracellular calcium, the KI,dark determination was accomplished with the DKO mice with the calcium-dependent feedback mechanisms knocked out. When the data from individual experiments (n = 9) were pooled together, the KI,dark obtained for the DKO mice was 15.0 ± 1.1 µM   Fig. . The KI,dark from the collected cGMP clamp data. Figure dark values were obtained from the slopes of the fitting of a linear fits to the plotted cGMP clamp data. The βdark values were 3.87 ± 0.04 s−1 for WT, 4.51 ± 0.09 s−1 for GCAPs−/−, and 4.40 ± 0.04 s−1 for DKO mice (mean ± SER). With DKO mice, local ERG was recorded across the rod outer segment layer, while with WT and GCAPs−/− mice the LERG recordings were conducted across the whole photoreceptor layer to improve the signal-to-noise ratio. The reason for using LERG-OS with the DKO mice was that the flash response data from the same experiments was used for modelling of flash responses (see below). Our results indicate that there is no significant difference in the βdark values between the tested mouse strains.After specifying the parameter values needed in equation , we deteR value in the vicinity of the optimum will change the βdark value determined from cGMP clamp, because βdark is proportional to A and τR . The deR,i, Ai, βdark,i) can describe the rod flash response behaviour. We modelled the fractional dim flash responses of GCAPs−/− and DKO mice, where the fractional responses were obtained by dividing the dim flash response with the saturation amplitude and the flash strength to give estimates for the sizes of fractional single photon responses. With GCAPs−/− and DKO mouse strains, the dim flash responses are not significantly modulated by calcium27. Therefore, we can apply equation . The insets in Fig. dark value and not the conjoined τR and A. With both mouse strains, the optimal βdark value coincided with that determined by cGMP clamp.To estimate which of these parameter combinations are plausible, we tested how well prospective parameter triplets (τequation  to modeldark), together with guanylate cyclase activity (α), determines the turnover rate and the intracellular concentration of cGMP in photoreceptors in darkness. Rieke and Baylor concluded that the natural variation in basal PDE activity in toad photoreceptors is the main source of the fluctuations in the intracellular cGMP concentration in darkness4 and that βdark is a key factor in setting the rod sensitivity and the temporal resolution of rod signalling in the dark adapted state. Still, no one has determined the rate constant for spontaneous cGMP hydrolysis in the small mammalian wild type photoreceptors. A pioneering effort to this direction was taken by Gross, Pugh, and Burns (2012), whose analysis was based on the assumption that the time constant determining the very latest recovery phase of dim flash responses is set by βdark in conditions, where the other time constants of phototransduction deactivation (τR and τE) are short compared to 1/βdark, and where there is no calcium feedback to response recovery5. These circumstances can be obtained in GCAPs−/− mice when τE is significantly shortened by overexpressing the RGS9 complex. Based on the time constant of the final recovery phase of dim flash responses, they estimated βdark to be 4.1 s−1 in GCAPs−/− background. However, the method is not valid for WT mice, in which the calcium-mediated feedback to guanylate cyclase activity accelerates the response recovery. For comparison, the basal PDE activity estimates vary from 0.1 s−1 to 1.5 s−1 in toad rods34, from 0.49 to 3.4 s−1 in frog rods35, and from 1.2 to 2.8 s−1 in salamander rods13 at room temperature.The basal PDE activity  or the time constant for deactivation of rhodopsin activity (τR). The ncGMP value is generally accepted to lie close to 3 for mouse rod photoreceptors5 and this value was used in the analysis. Larger uncertainty arises from the determination of τR. When analysing the lifetime of activated rhodopsin and the amplification constant A by modelling the early onset phase of flash responses, A and τR appear mutually dependent: a change in either of the parameter values requires an opposite shift in the other to achieve a reasonable fit . To obtain further support for our βdark determination by cGMP clamp, we modelled dim flash responses recorded from GCAPs−/− and DKO mouse retinas while restricting the parameter combinations to those that are plausible according to the activation phase modelling and cGMP clamp results . In our simultaneous determination of activation constant and rhodopsin lifetime  and for GCAPs−/− (4.5 s−1) mice do not seem to differ significantly. This is somewhat presumable because the expression level of PDE or the dark current level is not expected to change due to these genetic modifications43. Further, these values are in good agreement with that determined earlier for GCAPs−/− mice with a completely different approach (4.1 s−1)5. Therefore, it seems very reliable to conclude that the βdark value for WT mouse rods (as well as for GCAPs−/− mice rods) is close to 4 s−1. On the other hand, it has been proposed that the basal phosphodiesterase activity in mouse rods might be directly modulated by recoverin44. Our results suggest that removal of GCAPs (in the presence of recoverin) or recoverin (in the absence of GCAPs) do not significantly affect βdark. However, the essential feature of our cGMP clamp method is that the cGMP and Ca2+ levels are maintained at their dark adapted levels and therefore our results do not rule out the possibility that recoverin might modulate βdark as a result of changes in the intracellular calcium concentration. Furthermore, the novel method will allow determination of βdark value in cones, which likely differs from that in rods45, as well as investigation of potential modulation of βdark by factors such as glutamic acid-rich protein-246 or intracellular calcium concentration15. Overall, the introduced method offers a means to examine βdark in different animal models by recording ERG signals from the isolated retina, a situation closely resembling to in vivo condition.As illustrated in Fig. The use and handling of the animals were in accordance with the Finnish Act on Animal Experimentation 2006 and guidelines of the Animal Experiment Board in Finland. Experimental protocols were approved by Laboratory Animal Centre of the University of Helsinki, and by the Animal Experimental Board in Finland. In addition, Board for Gene Technology in Finland has approved our laboratory for the use of gene-manipulated mice.−/−, and GCAPs−/− recoverin−/− double knockout (DKO) mice, derived from GCAPs−/− and recoverin−/− mice42 kindly provided by Dr. Jeannie Chen , were used in this study. Mice were dark adapted overnight and sacrificed by CO2 inhalation followed by cervical dislocation. The eyes were enucleated and small incisions were made along the equators of the eyes. The eyes were bisected by enlarging the incision with micro scissors and the isolated eyecup was placed into cooled nutrition medium . One eyecup was stored at +7 °C in nutrition medium in a light tight container to be used later during the same day. The retina was removed from the eyecup with forceps and micro scissors under a microscope and the whole retina was placed in a specimen holder into a light-tight Faraday cage. The procedures described above were completed under a dim red light.Wild type (C57BL/6J), GCAPs47. Local electroretinography (LERG) were recorded across the rod outer segment layer (LERG-OS) or across the whole photoreceptors (LERG-PR). The LERG-OS signal is directly proportional to the changes in the rod outer segment current47. The recording electrode (with a tip diameter ∅ of 2–5 µm) was passed to the depth of ~25 µm (OS) or ~100 µm (PR) in the retina and the reference electrode (tip ∅ ~ 30 µm) was located on the surface of the retina. The surface was identified both visually and from the voltage shift observed when the recording electrode penetrated the surface of the retina. During the LERG recordings, simultaneous transretinal ERG recordings were conducted with macroelectrodes located on both sides of the retina. The recording geometry is described more in detail in47.Our specimen holder allows simultaneous visualization, stimulation and perfusion of the retina, and it is equipped with an open passage for microelectrodes enabling local electroretinography recordingca. 3 ml/min). The composition of the nutrition medium was (mM): Na+, 133.4; K+, 3.3; Mg2+, 2.0; Ca2+, 1.0; Cl−, 143.2; glucose, 10.0; EDTA, 0.01; HEPES, 12.0, adjusted to pH 7.5 with 5.8 mM NaOH. The viability of the retina was improved by adding 0.72 mg/ml Leibovitz culture medium L-15 to the solution. Synaptic transmission from photoreceptor cells to bipolar cells was blocked by adding 2 mM sodium aspartate48. To abolish the glial component arising from Müller cells, 50 µM BaCl2 was added to the nutrition medium49. 3-isobutyl-1-methylxanthine (IBMX) was used as a PDE inhibitor at concentrations of 5, 10, 20 and 40 µM. All chemicals were purchased from Sigma-Aldrich .The open chamber of the specimen holder was filled with nutrition medium and a laminar flow of medium perfused the photoreceptor side of the retina with a constant rate . Recordings were conducted at physiological temperatures 37 ± 1 °C. The temperature in the nutrition medium close to the retina was monitored continuously with a calibrated thermistor .max = 532 nm; Lumileds, Amsterdam, Netherlands). The stimuli illuminated the whole retina homogeneously as verified with a camera-based beam profiler (Spiricon Laser Beam Diagnostics Model SP503U). The absolute light intensity incident on retina was measured with a calibrated photodiode . The number of rhodopsin isomerizations (R*rod−1 or R*rod−1 s−1) caused by the stimulus was calculated based on the LED and photodiode spectra, respectively, and the pigment template by Govardovskii et al. (2000) as described in Heikkinen et al.50.Light stimulation was accomplished with 1 ms flashes from a LED light source  controlled closed loop feedback from the recorded ERG voltage signal to the light source was developed in order to keep the recorded signal constant when the PDE inhibitor was introduced to the retina. The light control feedback was carried out digitally with LabVIEW .c = 500 Hz (8-pole Bessel filter) and afterwards digitally at fc = 100 Hz.Data acquisition and LED controls were handled with a data acquisition card  and a custom made LabVIEW software. The recorded DC signal was sampled at 1000 Hz with a voltage resolution of 15 nV and amplified 1000-fold. The signals were low-pass filtered at f2.The following section introduces the theoretical background of the cGMP clamp method with help of phototransduction equations. The analysis is based on the phototransduction models described thoroughly inR* is the number of active rhodopsin molecules, PDE* the number of active PDE subunits, ΦBG is the number of activated rhodopsin molecules per second in one rod by the background light [ΦBG] = R*rod−1 s−1, kR and kE are rate constants for rhodopsin and PDE deactivation. α is the rate of cGMP synthesis and β is the rate constant of cGMP hydrolysis. βdark and βlight are the hydrolysis rate constants of cGMP by basally active and light-activated PDEs, respectively. βsub is the hydrolysis rate constant for one PDE* subunit and νRE is the rate constant for PDE activation by activated rhodopsin.In background light, phototransduction reactions can be assumed to obey first order reaction kinetics:In a constant background light, when the phototransduction reactions have reached a steady state, the concentrations of phototransduction molecules are invariant:β to βI according toβI is the hydrolysis rate constant of cGMP in the presence of the PDE inhibitor, [I] is the concentration of the inhibitor, k([I]) is a function describing the effect of inhibitor and KI is the inhibition constant for the inhibitor.The introduction of a competitive PDE inhibitor lowers the average hydrolysis rate constant of cGMP from PDE inhibitors decrease the hydrolysis rate of cGMP whereas light increases it. The decrease in the basal PDE activity due to the inhibitor can be compensated for by increasing light. For a steady state in the absence of light and inhibitor,In the presence of PDE inhibitor and light,βdark can be obtained by setting the cGMP concentrations equal in Eqs A is defined asncGMP = 3 is the Hill’s coefficient5 representing the cooperativity of the cGMP binding sites in the CNG channels21. From here we can simplify the equation  differ, the equation  decays according to first-order reaction kineticsΦ is the number of activated rhodopsins produced by the stimulus flash and τR is the average lifetime of activated rhodopsins. The calcium binding protein, recoverin, alters the τR in a calcium-dependent manner55. The activated rhodopsins can activate G-proteins, transducins, they encounter. An activated tranducin (α-subunit) can bind to a PDE molecule and activate it. PDE activation can be assumed to decay with first-order reaction kinetics and thus, the PDE activity can be solved from a convolutionνRE is the rate constant of PDE* formation by an activated rhodopsin and τE is the average lifetime of activated PDE. The rate constant of cGMP hydrolysis is determined byWe start with the simplifying assumption that after a stimulus impulse, the amount of activated rhodopsin . Therefore,−/− mice, the Ca2+-dependent modulation of cGMP synthesis is removed and Δα = 0. In GCAPs−/− recoverin−/− mice, also the calcium-mediated modulation of rhodopsin lifetime can be disregarded. With the DKO mice, equation cGMP(t)) which surpasses the combined effects from the rate of steady state cGMP synthesis (αdark = βdarkcGMPdark) and basal rate of cGMP hydrolysis (−βdarkcGMP(t)). This can be formulated mathematically as: βlight(t)cGMP(t) ≫ βdark(cGMPdark − cGMP(t)). Applying the relation to the equation  follows Ohmic relation with the voltage drop in the extracellular space across the rod outer segments, r(t), which is the signal registered in LERG across the outer segment layer (LERG-OS)56. The LERG-OS signal normalized by the saturation level, rmax, can be considered to followHere equation  simplifi−/− and DKO mice. In addition, we determined the amplification constants and rhodopsin lifetimes for WT, GCAPs−/− and DKO mice by fitting the model exploiting the equation (In this model, Eqs equation  to the b


GJB2 (connexin 26) and GJB6 (connexin 30) mutations associated with familial non-syndromic childhood hearing impairment (HI) in Cameroon. We selected only families segregating HI, with at least two affected individuals and with strong evidence of non-environmental causes. DNA was extracted from peripheral blood, and the entire coding region of GJB2 was interrogated using Sanger sequencing. Multiplex PCR and Sanger sequencing were used to analyze the prevalence of the GJB6-D3S1830 deletion. A total of 93 patients, belonging to 41 families, were included in the analysis. Hearing impairment was sensorineural in 51 out of 54 (94.4%) patients. Pedigree analysis suggested autosomal recessive inheritance in 85.4% (35/41) of families. Hearing impairment was inherited in an autosomal dominant and mitochondrial mode in 12.2% (5/41) and 2.4% (1/41) of families, respectively. Most HI participants were non-syndromic . Four patients from two families presented with type 2 Waardenburg syndrome, and three cases of type 2 Usher syndrome were identified in one family. No GJB2 mutations were found in any of the 29 families with non-syndromic HI. Additionally, the GJB6-D3S1830 deletion was not identified in any of the HI patients. This study confirms that mutations in the GJB2 gene and the del(GJB6-D13S1830) mutation do not contribute to familial HI in Cameroon.This study aimed to investigate  Hearing impairment (HI) is a disabling congenital disease with the highest rate for age-standardized disability of life in the world . GloballGJB2 (encoding connexin 26) and GJB6 (encoding connexin 30) as the major contributors to autosomal recessive NSHI (ARNSHI) [GJB2-c.35delG being the most prevalent variant (20–50%) found in cases of autosomal recessive non-syndromic hearing impairment (ARNSHI) [GJB6-D13S1830 deletion was identified in up to 9.7% of cases, and thus is the second largest contributor to the genetic etiology of NSHI in European populations, either with homozygous presentation, or when present in addition to a GJB2 mutation on the opposite allele [Non-syndromic hearing impairment (NSHI) is an extremely heterogeneous trait, with approximately 170 NSHI loci and 112 genes identified to date . Neverth(ARNSHI) , with GJ(ARNSHI) . The GJB2, with a 2017 census reporting a population of 24,053,727 [Cameroon is a sub-Saharan African country, covering an area of 475,442 km,053,727 . Cameroo,053,727 , and its,053,727 .GJB2 and GJB6 genes to HI in Cameroon [GJB2 and GJB6 genes to HI in Cameroon by only focusing investigations into cases that showed a clear pattern of inheritance within families.Previous studies have found no contribution of the Cameroon ,17,18. HThe study was performed in accordance with the Declaration of Helsinki. Ethical approval was obtained from the Institutional Research Ethics Committee for Human Health of the Gyneco-Obstetric and Pediatric Hospital of Yaoundé, Cameroon (No. 723/CIERSH/DM/2018), and the University of Cape Town’s Faculty of Health Sciences’ Human Research Ethics Committee (HREC 104/2018). Written and signed informed consent was obtained from all participants who were 21 years of age or older, and from parents or guardians in cases of minors, with verbal assent from participants, including permission to publish photographs.Hearing impaired patients were recruited from eight of the ten administrative regions of Cameroon, from schools for the deaf, and in the community, following procedures previously reported in Cameroon . Briefly® ), at the Biochemical Laboratory of the Centre Pasteur du Cameroun, Yaoundé, Cameroon, or using the Chemagic extraction protocol, in the division of Human Genetics, University of Cape Town, South Africa.Genomic DNA samples were extracted from peripheral blood, following the manufacturer’s instructions for the available commercial kit  and other software, as recommended [GJB2 gene (exon 2) was amplified, followed by sequencing using an ABI 3130XL Genetic Analyzer® , in the Division of Human Genetics, University of Cape Town, South Africa.Previously reported primers for the ommended . The entGJB6-D13S1830) was performed using the method and primers described by del Castillo et al. [GJB6 was amplified using the method described by Chen et al. [Detection of del years. The male/female ratio was 0.82 (42/51). Hearing impairment was congenital in 62 patients (66.7%), and the mean age at medical diagnosis was 3.2 ± 3.3 (1–22) years.Pure tone audiometry was performed in 54 of our 93 patients. Hearing impairment was sensorineural in 51 out of 54 (94.4%) patients and mixed in 3 patients; no patients exhibited a conductive hearing impairment. All of our patients had bilateral hearing impairment, and the majority had profound to total hearing impairment  .Pedigree analysis suggestive of autosomal recessive inheritance was the most frequently observed pattern of inheritance and accounted for 85.4% (35/41) of families A. In 12.The majority of our participants exhibited non-syndromic hearing impairment, which accounted for 92.5% (86/93) of cases, for a total of 38 families .Four patients from two families in our cohort presented type 2 Waardenburg syndrome (without dystopia canthorum). The main clinical signs included: hearing impairment, patches of hypopigmented skin, sapphire-blue eyes, and premature white hair .Three cases of type 2 Usher syndrome (without vestibular areflexia), from one family, were identified; in addition to hearing impairment, clinical signs of retinitis pigmentosa were present, including night vision impairment and constricted visual field.GJB2 and for the del(GJB6-D13S1830) mutation. None of the families exhibited the del(GJB6-D13S1830) mutation, or any of the reported disease-causing mutations in GJB2. However, a GJB2 variant of uncertain significance, NM_004004.6: c.499G>A (p.V167M), was present in one family in the heterozygous form  was shown to be highly prevalent in that population [GJB2 variant, c.499G>A (p.V167M), of uncertain significance according to the ClinVar database, was present in a family in our cohort, and has previously been described by our group in the Cameroonian population [GJB2 variant c.499G>A (p.V167M) could thus be considered to require further investigation.The low implication of the ucasians ,22,23, wucasians ,25,26,27deafness , and onlss [GJB2 . Howeverpulation , and accpulation . The GJBpulation . It has pulation  and in Cpulation . The GJBGJB6 is a 342-kb deletion, GJB6-D13S1830, which causes non-syndromic HI when homozygous, or when present on the opposite allele to a GJB2 mutation [GJB6-D13S1830) mutation is the second most frequent genetic cause of non-syndromic prelingual hearing impairment in the Spanish population (after the 35delG mutation in GJB2) [GJB6-D13S1830), the coding region of GJB6 was sequenced in African probands from Cameroon, South Africa, and Uganda; however, this revealed no additional pathogenic mutations [The most common mutation in mutation ,34. The in GJB2) . This dein GJB2) ,35,36, bin GJB2) ,39,40,41in GJB2)  and in Gin GJB2) . In ordeutations .GJB2 and GJB6 do not play a significant role in non-syndromic hearing impairment in most populations of African descent. Interestingly, genetic testing through targeted genomic enrichment and massively parallel sequencing of 116 genes were used to screen 10 multiplex families with non-syndromic hearing impairment. In 7 of the 10 families (70%), 12 pathogenic variants were identified in 6 genes, and nearly half of these variants were novel [Our findings support previous reports that re novel . Therefore novel , and allre novel .The study also confirms Waardenburg syndrome as the most common cause of syndromic hearing impairment in Cameroon, in line with previous reporting in other African populations ; these fGJB2 gene and the del(GJB6-D13S1830) deletion are not implicated in familial non-syndromic hearing impairment in Cameroon. Future studies should employ whole genome sequencing approaches and functional genomics studies to identify other genes that may be implicated in the hearing impairment observed in these families.The present study showed that hereditary hearing impairment in Cameroon is mostly non-syndromic, congenital, sensorineural, and inherited in an autosomal recessive mode. Additionally, this study identified Waardenburg and Usher syndromes as the most common syndromic hearing impairments in Cameroon. This study confirmed that mutations in the


A patient-orientated approach in medical clinical practice is emerging where patients and practitioners are considering and including the spiritual, emotional and psychosocial aspects of the individual. This practice is an important change in health care, specifically in the field of audiology as a holistic view of the patient now alters the perspective on the management of individuals with hearing impairments.This article explored the experiences of a participant who reported supernatural healing of his sensorineural hearing loss (SNHL). Hence, this study focuses on the consideration of spirituality in the inclusive model of care.An exploratory, qualitative narrative inquiry was used to obtain data from a single pilot case study of a 27-year-old man who reported healing of his permanent profound hearing loss.Four themes were identified within the narrative obtained: prayer and faith, deaf culture, identity and purpose. The participant stated that he believed that he was partially healed to fulfil his purpose in life. The partial healing allowed him to belong to the deaf community and the hearing world simultaneously.South Africans live in a diverse society where most people accept spirituality as part of their search for meaning in life. Health care for individuals should therefore consider the person as a holistic being more than a medical entity. The exploration of narratives of individuals who report supernatural healing of a SNHL will assist health care practitioners and audiologists in managing individuals in an inclusive manner. This pilot study thus has implications for policy and practice in health care contexts. Permanent cochlear damage resulting in a SNHL may be associated with presbycusis, ototoxicity, injury, trauma, diseases and congenital aspects Møller, , with irWhat can be lost that was never there in the first place?When considering changes in health care in relation to audiology, the impact of mental health issues should be included. The current study does not directly focus on mental health issues; however, they are included in the biopsychosocial–spiritual model adopted. There is very little international research relating to alternative methods to manage a hearing loss. The individual’s approach to health care is shaped by their religious and cultural practices. Culture plays a significant role in the way disability is viewed by an individual  their religious beliefs and practices, and a person’s faith. These contribute to the biggest influences in that individual’s reactions to life events, including illness and disease. Spirituality is one of the key strengths in personal well-being  the supernatural intervention and healing process .A single pilot case study was conducted with a 27-year-old South African man, who reported experiencing supernatural healing of the hearing in one ear at the age of 7. The participant was diagnosed with a profound bilateral SNHL at birth and attended a school for deaf children. The participant stated that he was diagnosed and fitted with bilateral hearing aids; however, he did not use the one hearing aid post-healing. The focus of this study was to document the participant’s experiences of the ‘healing’ in relation to the hearing loss. The study did not aim to prove that the hearing loss was ‘cured’; therefore, a new audiogram was not required as proof of the recovery of hearing.The participant is of the Christian faith and he reported that the hearing in his left ear was healed, and his healing, he believed, was an answer to the prayers of his family:‘I was six years old or seven years old … I started to hear on the right, no on the left ear (pointing to the left ear). So far I’m happy, I’m Christian, and I know God always helps me to hear. Maybe later on He (God) will heal me 100%, because now I’m growing up now I know sign language and I’m English speaking. And I believe God helped me to speak and to hear. Even though I cannot hear in right ear, nothing in the right ear, only the left ear. I can speak, show the people how I talk, how I prove how I feel God’s presence.’ ‘So I understand different situations, I’m happy with God, I always pray. My family they (are) Christians, my uncle and my cousin are pastors. I have another uncle that is a pastor too. There are so many people who are Christian in my family. And I realised that they were praying for me, God answered their prayers to improve my life and I received lots…’ From this narrative, the importance of identity with the deaf is evident. This participant holds a strong deaf identity, as he relates, communicates and identifies with the deaf culture; however, he also continues to see himself as a hard-of-hearing individual. He stated that he feels:‘God helped me to hear partially because there’s so many Deaf people who don’t know God and so God was giving me purpose, to communicate with Deaf people and hearing people, that’s what I feel. So I share the Word of God, about God with the people whose hearing and Deaf. So far I’m happy that I still stayed hard-of-hearing because I can communicate with this side and this side (meaning with the hearing and the hearing impaired). If, imagine if God healed me 100% full I wouldn’t have talked with Deaf people about God, cos they don’t know. I feel God’s purpose for me to be hard-of-hearing and I’m happy with who I am. So I’m happy that I’m hard-of-hearing and I can hear a little bit and I know Deaf Culture, Deaf feelings, how the people feel if they don’t hear and how the people who hear, how they feel. So I understand different situations, I’m happy with God, I always pray.’ This participant’s identity is driven by his ability to define this healing event as a purpose-driven event in his life.Human interaction and relationship-building dictate that it is inevitable that there will be questions surrounding such claims of healing. This participant had a friend of a different faith, who questioned his healing and the mechanisms behind it as the friend was curious about the reasons for the healing.‘My one friend is Muslim, he don’t believe in God, how I believe, how I, he asked where my hearing comes from. Maybe spirit, coming from God? People they can’t see God, the spirit, the relationship, the feeling, even hearing from God is a must. But must get the information in the heart, in my mind when you read the Bible, when you ask something to God and you are rushing to get the answer, one day when I was reading the Bible, that’s the answer (pointing to the page in a book). Then I see the Bible is the truth and now many people don’t believe the Bible, (they ask) who wrote the Bible, how he gets the words from? And I try to explain, people who wrote because they get their message from God, through hearing, dreams…’ From the results of the participant in the pilot study, several themes have emerged that confirm or support findings discussed in the literature. The following four themes are discussed below: prayer and faith, deaf culture, identity and purpose.laying on of hands  specialist within the multidisciplinary team. The ENT can use the information obtained by the audiologist to benefit his or her own assessment and management practices. If we believe that the medical clinical model is failing the patient , then we need a shift in mind-set. This shift means that audiologists need to be moving towards a goal of including questions relating to faith and spiritual beliefs from the initial consultation with the patient.Deaf culture, identity and purpose are themes that conjure up further questions for us as audiologists. Are we defined by the illness or disease? Who are we and why are we experiencing these situations in life? Are we designed to teach others about perseverance as part of the process of overcoming difficult situations?The purpose of life is an ongoing enigma for every individual. People tend to rationalise good and bad events so that it allows them the ability to accept the situation at hand and move on. The 27-year-old participant in this study referred to his deafness as a positive factor in his life as he stated that he believes that he can communicate with the deaf, and impact them by sharing information about God. His purpose was defined as the provider of life-changing information to the deaf. The participant stated that he was bilingual as he is proficient in both sign and oral language. Furthermore, he sees himself as a bridge between the deaf and hearing worlds. He reiterates that there is a reason for his partial hearing as he now belongs to a ‘bicultural’ world, straddling the hearing and deaf communities allowing him to share his knowledge about God. Cultural and linguistic factors are therefore important considerations in medical clinical management.Similar to the participant in the current study, research has indicated that long-term breast cancer survivors reported a deeper spiritual experience following their diagnosis (Sabado & Tanjasiri, Change is required from a medical clinical model to a more inclusive model of intervention within health care and this has been recommended in the literature. Increased reports of more ‘personal experience evidence’, is, however, now available to support transformation in intervention approaches. This study therefore argues for an inclusive approach to intervention in audiology.We live in a spiritual society and most people accept a spiritual aspect in their lives as they seek to obtain a meaning of life Bandura, . The medThe medical model has led to the person being seen as a specimen and despite the advances in cracking the genetic code, the understanding of the human as a whole has stagnated Sulmasy, . As far Sulmasy  supportsThe mediating structure of community is vital as the community is a source of social resource and social identity (McLeroy et al., The following questions are relevant and should be reflected on to create the shift in health care practice:How do we transition from the old to the new inclusive health care process?Are audiologists equipped and ready to change the way we assess and manage our patients?Audiologists are making progress and the evidence must be documented in further studies within the areas of supernatural healing and medicine. This study should therefore contribute further to understanding the changes needed in medical clinical practice.


To explore the correlation between hearing and speech recovery levels after cochlear implantation and examined the preoperative microstructure of auditory pathways and speech centre using DTI.λ‖), radial diffusion coefficient (λ⊥), and MD values in the lateral lemniscus, inferior colliculus, medial geniculate bodies, auditory radiations, Brodmann areas 41, 42, 22, 44, 45, and 39 were all measured bilaterally. (2) CAP and SIR scores were assessed in fourty-six cochlear implantation children at 6 months post-implant. Correlations among deaf children ages, FA value of bilateral inferior colliculus FA values, BA22, BA44, and postoperative CAP, and SIR scores were analyzed using multiple linear regression.(1) Fifty-two SNHL children between 0 and 6 years and 19 age and gender matched normal hearing subjects had received 3.0 T-MRI examination of the brain.FA, axial diffusion coefficient  was slightly greater than that of the inferior colliculus (|bi′| = 0.377) FA value.Preoperative children ages and inferior colliculus FA values were important factors influencing postoperative CAP score. Inferior colliculus FA value is a vital influencing factor in rehabilitation after cochlear implantation. Cochlear implantation (CI) can help patients with severe and extremely severe deafness recover partial hearing. It is currently the only effective method to treat children with congenital severe and very severe sensorineural hearing loss (SNHL) . CochleaFifty-two congenital sensorineural deaf children aged 0.9–6 years, who were prepared for cochlear implantation in our hospital from May 2014 to March 2017 were the research subjects. Among the 52 selected subjects, 35 were males and 17 were females, with an average age of 3.32 ± 1.53 years. Inclusion of standards and exclusion criteria: (1) the child, at birth, had no hearing or extremely poor hearing, did not respond to outside general sound. This resulted in extremely severe sensorineural deafness in the pure tone hearing test > 90 dB; (2) After wearing an appropriate hearing aid, hearing and speech training did not achieve any effect after hearing rehabilitation training for 3–6 months. Not included were those with severe inner ear malformation, such as Michel malformation, auditory nerve deficiencies or interruption, serious mental, intellectual, behavioural, or psychological disorders, inability or unwillingness to cooperate with language trainers, and serious mental illness. In contrast, 19 age- and gender-matched children with normal hearing, age 1.17 to 6 years, formed the control group. The control group consisted of 13 males and 6 females, with an average age of 3.8 ± 1.58 years. Temporal bone and head images were normal, and there was no history of neurological disease and family history. The subjects were young and unable to complete the magnetic resonance (MR) examination in an awake state. Subjects were orally administered a 10% chloral hydrate dose of 0.5 ml/kg before the MR examination. Prior to the test, the guardians of all subjects provided signed informed consent. This study was approved by the Hospital Ethics Committee.2/s and 800 mm2/s; proliferation sensitive gradient direction = 32; scan time = 9:40.Cranial axial DTI scanning using a Philips Achieva 3.0T X-Series MR imager in single-shot planar echo imaging sequence (SS-EPI) with a head SENSE eight-channel phased-array coil was performed. The settings were: TR = 5090 ms, TE = 70 ms, layer thickness = 3 mm, layer spacing = 0, FOV = 220 × 220 mm, matrix = 160 × 160. Other conditions were as follows: excitation times = 1; scan layers = 45; b values = 0 mm0 images using FSL5.0 software. Eddy current correction and realignment were implemented. A fitting tensor model with the least square calculated the subjects’ FA, λ‖, λ⊥ and MD numerical map.Data analysis and processing: a dcm2nii software of MRIcron converted the collected original DICOM 3.0 format files into NIfTI format files. The processed data were registered to bλ‖, λ⊥ and MD for each subject. The ImCalc module of SPM8 software was run on a Matlab platform to calculate average FA, λ‖, λ⊥ and MD values for each subject.Extraction of region of interest (ROI): double inferior colliculus, medial geniculate body, BA41, BA42, BA22, BA44, BA45, and BA39 were extracted from a Brodmann template, which was included in PickAtlas software of Wake Forest University (WFU), as a mask. Lateral lemniscus and auditory radiation in a Brodmann template could not be extracted as a mask in this study. The average FA map of the three groups’ subjects after registration was used as a template to sketch lateral lemniscus and auditory radiation information areas. These were added to the Brodmann template , radial diffusion coefficient (λ⊥) and mean diffusion (MD) for each subject’s lateral lemniscus, inferior colliculus, medial geniculate body, auditory radiation, BA41, BA42, BA22, BA44, BA45 and BA39 were obtained , axial diffusion coefficient (y), and age (x1), inferior colliculus FA value (x2), BA22 FA value (x3) and BA44 FA values (x4) as corresponding independent variables. The CAP multiple linear regression analysis results of 46 SNHL children appear in Table CAP- and SIR-based multiple linear regression analysis: CAP and SIR scores were assigned by auditory appraisers to the 46 SNHL children. A multiple linear regression analysis was performed on the CAP and SIR scores after the CI operation using dependent variables  is no hearing, or very poor hearing, at birth. CD neonates had no response to general public voice. Most were diagnosed as SNHL. CD incidence among neonates was approximately 1–3% . SaksenaThe human auditory conduction pathway consists of a sensory part and a neuro part. The neuro part involves multiple neuroanatomical structures, including the vestibular cochlear nerve, cochlear nerve nucleus, trapezoid body, superior olivary nucleus, lateral lemniscus, inferior colliculus, medial geniculate body and an auditory centre . A speecλ‖, λ⊥ and MD value changes were of statistical significance.At present, diffusion tensor imaging (DTI) is the only non-invasive detection method capable of showing the microstructure and pathological status of white matter fibre tracts in vivo . Recentlλ‖ and MD values of the deaf group were lower than that of the control group. Deaf group λ⊥ values were higher than that of control group  of pre-operative deaf children’s ages was slightly larger than that of the inferior colliculus FA value (|bi'| = 0.377), indicating that the impact of pre-operative inferior colliculus FA values on post-operative cochlear implantation effect was equivalent to that of deaf children’s age. The reason may be that the inferior colliculus is a third-level neuron of the auditory pathway, where auditory reflex synapses are located, and where many upper and lower fibre bundles of the auditory pathway gather. Figures This study has some limitations. First, the follow-up time for this study was only 6 months post-CI. At present, it is thought that auditory and verbal recovery tends to stabilise at 12 months post-CI . Correla


The high sound pressure level generated by impulse noise produced in an indoor shooting range makes it necessary to protect the hearing of the people it affects. Due to the need for verbal communication during training at a shooting range, level-dependent hearing protectors are useful. The objective of this study was to answer the question of whether it is possible to properly protect the hearing of a shooting instructor using level-dependent hearing protectors. The noise parameters were measured in the places where the instructor was present at the shooting range. The division of a specific group of trained shooters into subgroups consisting of three or six simultaneously shooting individuals did not significantly affect the exposure of the shooting instructor to the noise. An assessment of noise reduction was carried out for eight models of earmuffs and two variants of earplugs, using computational methods for the selection of hearing protectors. Among the noise parameters, both the A-weighted equivalent sound pressure level and the C-weighted peak sound pressure level were taken into account. Depending on the assessment criterion adopted, a sufficient reduction in impulse noise was provided by either four or six out of the 10 hearing protectors included in the study. Impulse noise is particularly dangerous for hearing due to its fast-changing nature. A single exposure to noise produced from an artillery shot can lead to a temporary hearing loss, while repeated exposure to noise associated with gunfire can result in permanent noise-induced hearing loss . AuditorThe need for communication in the presence of noise, in terms of the use of hearing protectors, is becoming increasingly common in many workplaces . For noiRegardless of the aforementioned functionality of level-dependent hearing protectors in terms of support in the perception of ambient sounds, these protectors should limit exposure to noise in such a way that the hearing of their users is safe. Therefore, it is necessary to properly select hearing protectors on the basis of the noise parameters present in the location of the user of the hearing protectors. The selection of hearing protectors can be carried out using computational methods. It is then necessary to determine both the noise parameters present in the workplace and information about the sound attenuation of the hearing protector in question. The selection of hearing protectors is not often discussed and, for example, is limited to presenting the basic principles of such a selection or discussing the main problems involved ,11. It iIt is not difficult to obtain information about the sound attenuation of hearing protectors used in computational selection methods for continuous noise. This attenuation is one of the parameters measured during the conformity assessment process for the EU regulation , and itsThe objective of this study was to answer the question of whether it is possible to properly protect the hearing of a shooting instructor at an indoor shooting range, using level-dependent hearing protectors. The study includes a large number (10) of level-dependent hearing protectors. In addition, the assessment of impulse noise reduction was carried out not in the presence of laboratory-produced acoustic impulses, as in the method described in ANSI/ASA S12.42-2010  , but insLevel-dependent hearing protectors were included in the assessment of the protection of the hearing of a shooting instructor. There were eight different models of earmuffs and two variants of earplugs with two types of eartips made of polyurethane and silicone, respectively. The use of the two types of eartips is associated with different sound attenuation values. Therefore, in this study, both variants of eartips are considered as two separate hearing protectors. In Measurements of noise parameters were carried out during shooting exercises at the acoustically treated indoor shooting range, the dimensions of which were 35 m in length, 8.5 m in width and 2.6 m in height. As the possibility of protecting the shooting instructor’s hearing was being assessed, noise measurements were carried out in the places where the shooting instructor was present during the training, with the instructor at three different distances from the shooters: 1.1 m, 2.8 m, and 16.5 m .Glock 17 pistol  and Walther P99 , hereinafter referred to as the ‘pistols’;PM-98 Glauberyt submachine gun , hereinafter referred to as the ‘submachine gun’;Mossberg 590 smooth-bore shotgun , hereafter referred to as ‘shotgun 1’;Mossberg 590 smooth-bore shotgun , hereafter referred to as ‘shotgun 2’.The measurements were carried out during training, which consisted of four types of shooting cycles, during which shots were fired with the following weapon/ammunition combinations:The measurements included a total of 53 shooting cycles, of which 17 cycles were shot from pistols, 16 cycles were related to shots from a submachine gun, 10 cycles were shot from (smooth-bore) shotgun 1 and 10 next cycles were shot from (smooth-bore) shotgun 2.The training took place with two variants of the number of shooters simultaneously shooting during the shooting cycle: three or six shooters. The duration of one shooting cycle was 300 s (three shooters) or 420 s (six shooters). The duration of one shooting cycle means a full representative cycle of the task, determined in accordance with the principles for noise measurements at the workplace specified in the standard EN ISO 9612 . The cycSPL in octave bands with center frequencies: 125 Hz, 250 Hz, 500 Hz, 1000 Hz, 2000 Hz, 4000 Hz, 8000 Hz.Aeq),A-weighted equivalent SPL (LCeq),C-weighted equivalent SPL (LCpeak).C-weighted peak SPL  [EX,8h is based on LAeq, taking into account the time of exposure to noise Due to the fact that, in the assessment of noise exposure at the workplace, both the parameter related to the impulse nature of noise, L ,19), areISO 9612 .Cpeak or LEX,8h, is exceeded. For the LCpeak parameter, Directive 2003/10/EC [EX,8h parameter, in accordance with the provisions of the Directive [EX,8h at 89 dB can be supplemented since one of the papers concluded that, in the case of impulse noise [Noise is classified as dangerous to hearing when an exposure limit value of one of the parameters, i.e., L03/10/EC  sets the03/10/EC  was adopirective , should irective . There ase noise , this waIn this study, a G.R.A.S. 67SB  transducer was used for measuring noise parameters, which is designed for impulse noise parameter measurement . The uppIn accordance with the recommendations of EN ISO 9612 , the meaThe assessment of noise reduction by the hearing protectors included in the analysis was carried out by the so-called selection of hearing protectors. The selection consists of calculating the A-weighted SPL n EN 458  were usen EN 458 . The othstandard  and is eCalculations with the octave band method are made using sound attenuation data  in sevenCpeak in Equations (2) and (3) is the C-weighted peak SPL measured at the workplace.In the case of exposure to impulse noise, the assessment of noise reduction by hearing protectors is more complicated, because the instructions for the use of the hearing protector do not contain data related directly to impulse noise. Therefore, appropriately adjusted hearing protector data should be used; i.e., the previously mentioned H or M or L parameters. Due to the fact that a specific hearing protector limits the acoustic impulses generated by various sources to various degrees, these sources are divided into three groups , and the method of calculation depends on which type of source is considered. The assignment of the noise source to the noise type is related to the frequency range in which the dominant spectral components of the signal energy generated by the considered source are located. The impulse noise produced during shots from a pistol or a submachine gun is classified as type 3 noise. Then, the value of the The assessment of hearing protectors for their user’s hearing protection is based on a comparison of the calculated values otectors . Therefootectors , which aotectors , the LCp03/10/EC , after wStatistical analysis of the obtained measurement data was carried out in order to assess whether there were significant differences in exposure to noise resulting from the two variants of the number of shooters simultaneously participating in the exercises and to assess the impact of distance on the values of noise parameters. Data were analyzed using MATLAB R2017b (version 9.3) software with the Statistics and Machine Learning Toolbox . The analysis used the Wilcoxon test, equivalent to the Mann-Whitney U test.Aeq values measured for both variants of the number of shooters simultaneously shooting, with three different distances between the shooting instructor and the shooters. The highest LAeq values occur during pistol shots, and the lowest during shotgun 1 shots. The results of the measurements indicate a noticeable decrease in the LAeq value, associated with the increase in the distance of the instructor from the shooters. The decrease is about 3 dB if the distance is changed from 1.1 m to 2.8 m, and another 8 dB if the distance is increased to 16.5 m. However, the LAeq values shown in Cpeak parameter ; however, the deviations of results from the theoretical line are also relatively small (as in the case of LAeq parameter).Similar to the LEX,8h values are determined using Equation (1), taking into account the calculated time of exposure to noise, in the case of both variants of the division of shooters into teams. LCpeak values measured in analogous situations are also included.The influence of two different, practically applicable scenarios of conducting shooting exercises on the exposure of a shooting instructor to noise was analyzed. Usually, the task of the instructor is to carry out training with a group of 18 shooters in one day. The training may be conducted with a group divided into six three-person subgroups or three six-person subgroups, within which the shooters shoot simultaneously. In the case of three-person teams, the total time of exposure of the instructor to the noise is 1800 s (6 × 300 s), or 1260 s for six-person teams (3 × 420 s). In EX,8h, the variation of the value of this parameter related to a different number of shooters is not significant, and the average difference between the situation of three shooters firing in six subgroups and the situation of six shooters in three subgroups is 0.3 dB. It is worth noting that in most cases (16 out of 26), LEX,8h had a higher value for three-person teams than for teams of six people. The reverse situation concerned eight out of 26 cases. The value of LCpeak associated with the simultaneous firing of shots by three shooters in six subgroups in relation to the situation in which shooting takes place in three subgroups of six shooters, which is more negative than positive . On average, the difference in the LCpeak value between the two variants (three and six shooters) of the number of shooting teams is −0.8 dB.In the case of LCpeak and LEX,8h, there are no statistically significant differences between the results obtained with 3-person teams, compared to the results for 6-person teams . The conclusion which follows from the described analyses is that a different number of people simultaneously shooting (three or six) does not significantly affect the shooting instructor’s exposure to noise.On the basis of the results of the statistical analysis, it can be concluded that, both in the case of LCpeak and LEX,8h parameters, divided into three variants of the instructor’s distance from the shooters. Determined p-values for comparisons of the LCpeak and LEX,8h values corresponding to different distances of the shooting instructor from the shooters; i.e., for the comparison of results measured at 1.1 m and 2.8 m, at distances of 2.8 m, 16.5 m, 1.1 m and 16.5 m, were many times smaller than 0.05 in every situation compared. The results of measurements of noise parameters at different distances were therefore significantly different for individual distances. It has been confirmed, therefore, that the change in distance significantly affects the exposure of the shooting instructor to noise, due to both the LCpeak and the LEX,8h parameters.The positive effect of increasing the distance of the instructor from the shooters in order to reduce the exposure to noise is basically obvious. However, a verification was made to determine whether, considering the plan of exercises carried out at the shooting range, including the four weapons/ammunition combinations considered, the effects of increasing the distance of the instructor from the shooters should be treated as statistically significant. This verification was carried out with consideration of the parameters used in the noise exposure assessment criteria at the workplace. The results of the selection of hearing protectors with respect to the noise parameter associated with its impulse character, i.e., The selection of hearing protectors by the octave band method results in the obtained The highest absolute values of difference between the calculations of the The highest values of The research conducted indicated that the noise produced in individual training situations at the shooting range was different in terms of the content of acoustic energy components in individual frequency bands . Taking Cpeak, depending on the variant of weapon/ammunition combination and the shooting scenario, assumed values ranging from 145.9 dB to 158.1 dB. There are, however, no data that could be directly compared with the data obtained in this study at the indoor shooting range for the instructor’s locations. Published results usually characterize noise close to the shooter’s ear , or there is no precise information about the measurement location and C-weighted peak SPL or unweighted peak SPL (Lpeak) being considered; moreover, measurements are usually carried out on military fields. For example, in [peak associated with C7 rifle shots was 148.3 dB, while at the distance of 30 cm from the shooter, it was 154.7 dB [peak determined on the basis of waveforms associated with a 5.56 mm caliber C7 rifle shot, recorded at a distance of 4 m from the muzzle at 90° and 6.4 m at 39°, in both cases, the value was approximately 157 dB. In one of the studies, the Lpeak values associated with AR-15 rifle shots were 168 dB at the distance of 1.8 m, 150 dB at 4.3 m, and 132 dB at 25.7 m [The measurements of the impulse noise parameters generated in the places where the shooting instructor was located indicated relatively high SPL values produced by the firearms, which is in line with the results of other studies on shots from firearms. Measurements conducted in this study showed that the instructor, when standing at a distance of 1.1 m behind the shooters, was exposed to acoustic impulses whose L154.7 dB . For sho154.7 dB . Subsequ154.7 dB  indicatet 25.7 m .Aeq and LCpeak values shown in In one of the studies, where strategies for possible ways to reduce the negative impact of impulse noise on shooters’ ears were formulated, among others, the use of outdoor or acoustic-treated indoor shooting ranges was recommended . The resCpeak parameter, in this paper, for each hearing protector, two attenuation values were distinguished: one for shots from pistols and a submachine gun, and the second for shots from smooth-bore shotguns (shotgun 1 and shotgun 2). The different values of noise parameters produced by each weapon/ammunition combination and the different values of the acoustic parameters of individual hearing protectors resulted in different noise values determined under the hearing protectors. For example, the highest Cpeak value of noise is exceeded.In the case of impulse noise, it is not possible to characterize the reduction of this kind of noise by a specific hearing protector using one value only. This is due to the fact that the parameters of acoustic impulses produced by different sources differ from one another , and the′ values  under thpeak of 150 dB [peak to 108.8 dB. In similar measurement conditions in the next study, data were obtained that indicated that another earmuff reduced Lpeak to 118 dB [Cpeak value with the HP2 protector at distances from the shooter equal to 1.1 m and 2.8 m was 113.3 dB for submachine gun shots and 125.0 dB for shotgun 2 shots. Examples of published data can be supplemented with the result of 132 dB concerning the Lpeak parameter measured using an acoustical test fixture with an earmuff, in the presence of an impulse produced during a shot from a 12.7 mm machine gun [peak value of 152 dB. The studies in the referenced papers [There are no published data on the reduction of impulse noise by individual hearing protectors, which could have been directly compared with the results presented in this paper. For example, data are available for the insertion loss of impulses generated during shots from an AR-15 rifle with a Lf 150 dB . These df 150 dB  reduced o 118 dB . In thishine gun . The impd papers ,29 and tSince a hearing protector can only be considered suitable for protecting hearing against noise when its use obtains correspondingly reduced parameter At the same time, it can be noted that by changing the criterion for irective , an adeqCpeak, is crucial in the process of assessing hearing protectors in terms of their ability to reduce the impulse noise produced on a shooting range. In both situations in which the parameter Cpeak.The results obtained indicated that the parameter associated with the instantaneous value of the signal, i.e., LAt the same time, it should be noted that hearing protection requires not only properly selected hearing protectors, but that they must be used and set up correctly, and other sources of noise exposure, often unrelated to professional work, should be avoided. For example, practical instances of the improper use of earplugs were observed, and the reason behind this was a lack of training or that it was deliberately done to better hear messages spoken by the Range Safety Officer . In turnThe results of the study indicate that impulse noise produced on the shooting range should be assessed as dangerous for hearing. The exposition of the shooting instructor to this noise requires the use of appropriately selected hearing protectors. The results of the analysis of noise parameters measured at different distances of the shooting instructor from the shooters confirmed that increasing this distance, if possible, is a good method for reducing the exposure of the instructor to noise. It also appeared that with a certain number of shooters who are to be trained, their division into subgroups consisting of three or six people at the same time does not significantly affect the shooting instructor’s exposure to noise.The results of the assessment of level-dependent hearing protectors demonstrated that it is possible to adequately protect the hearing of a shooting instructor who is at an indoor shooting range when using protectors of this type. It is possible to choose hearing protectors, both earmuffs and earplugs, that will sufficiently reduce the impulse noise to which the instructor is exposed.It appears that, taking into account the program of exercises at the shooting range at which shooting was carried out with pistols, a submachine gun and smooth-bore shotguns, the assessment of hearing protectors with a more restrictive criterion