Behavioural and physiological markers of discrete positive emotions remain little investigated in animals. To characterise new markers in horses, we used tactile stimulations to induce emotional situation of contrasting valence. In the Gentle grooming group  horses were gently groomed during 11 sessions on the body areas they appreciated the most. Horses in the Standard grooming group  were groomed using a fixed procedure, reported to induce avoidance reactions in some horses. At session 11, G horses expressed significantly more contact-seeking behaviours than S horses, which expressed significantly more avoidance behaviours. This result suggests positive emotions in G horses and negative emotions in S horses. Blood cortisol, oxytocin, heart rate and heart rate variability never differed between before and after the grooming session. However, after the 11 sessions, basal oxytocin levels were lower in the G than in the S group. This difference was unexpected, but supports studies showing that a low level of basal oxytocin could be a marker of better well-being. Analyses of facial expressions during grooming revealed significant differences between groups. These expressions appear to be more sensitive than behavioural indicators because they alone enabled differentiating emotions according to the group when horses were re-exposed to neutral grooming one year after the treatment. Long restricted to negative emotions such as fear, present-day work increasingly focuses on the positive side of emotions3. One of the challenges of research in species which do not share verbal language like humans is to be able to characterise the type of emotion felt by a subject at a given moment. To this end, different kinds of indicators can be used3.The study of animal emotion is a field of research that has developed considerably in recent years. In the context of a ‘dimensional approach’, emotions can be characterised according to their two main dimensions4. In addition to body language, facial expressions, which are often automatic and very stereotyped5 are also well known to convey emotional information in humans and non-human primates. In humans, characteristic facial expressions have been correlated with specific emotions such as fear, anger, joy and surprise6. These expressions have been increasingly studied in animals, in particular in species such as primates, dogs and rodents, which like humans, have a large number of facial muscles (review7). They have been widely investigated for negative states such as pain  or fear (horses14) through grimace scales or during specific activities such as horses being ridden15. Facial expressions have also begun to be investigated for positive emotions. A recent study carried out on rodents identified facial indicators of positive emotions during playful manual tickling16. The authors revealed that the ears of the rats became more relaxed and pinker during tickling, compared to a negative situation such as exposure to a novel room and white noise. Other studies, particularly in cattle, sheep and horses analysed changes in certain features of the face such as eye shape18, wrinkled eyes19 or ear postures23. For instance, Proctor & Carder17 found that stroking, when perceived positively reduced the percentage of visible eye white in dairy cows. In horses, Hintze et al.19 reported that the angle between the line through the eyeball and the highest wrinkle decreased during grooming. Studying facial expressions thus seems to be an interesting lead to characterise emotions, and the horse is an appropriate model for at least two reasons. First, the horse benefits from complex face muscles that allow it to express a wide range of facial movements, including many that are also seen in primates24. Second, the horse is a social species that establishes sophisticated and long term social relationships, thus requiring effective communication. Given the fact that it is predominantly a visual animal, it is reasonable to think that it uses postural and facial expression to communicate with its conspecifics24. In particular, the literature describes a wide range of body postures and some facial expressions that are expressed in social contexts . A better understanding of horses’ facial expressions in connection to their emotions could be very helpful to develop new tools to assess and improve their welfare, a key societal challenge.The first category concerns behavioural indicators. Some of them can be a reliable source of information regarding the subjective experience of animals. In particular, reward-seeking and avoidance behaviours can notably inform us about the emotional valence of a stimulus26, horses28). However, little research has described the relationship between physiological responses and positive emotions. A few studies have reported an effect on heart rate parameters. In humans, Steptoe29 reported a lower heart rate in the case of positive affect. In horses, a decrease in heart rate has also been observed in positive contexts such as grooming30. RMSSD (Root Mean Square of the Successive Differences) have sometimes been considered as an indicator of emotional valence (e.g. in pigs31 or in sheep3). However, the major issue with these studies is that the effect of arousal was usually not controlled, thus it is not conclusive as to whether RMSSD were a marker of valence or of arousal. When these factors were controlled, as in the study of Briefer32, heart rate variability was affected by arousal and not by valence. At the hormonal level, cortisol has sometimes been described as being lower following positive situations . Rises in oxytocin levels have also been observed in positive situations, for instance following positive interactions between a dog and its owner33 or following a massage session in rats35 and humans36. Nevertheless, the findings are not always clear, with opposite effects being observed. For example, a study conducted in sheep showed that oxytocin levels did not rise after a positive contact with the animal caregiver, but did increase after the animal had been left alone37. Further research is thus required to deepen our understanding of physiological correlates of positive affective states.The second category of emotional indicators concerns physiological markers. These markers have been relatively well investigated regarding negative emotions. For example, rises in heart rate parameters or in cortisol levels have often been described in response to stress-inducing situations  or by positive responses such as contact seeking or a relaxed state . We thus compared two groups of horses groomed in a different manner for 11 sessions of ten minutes each. The Gentle grooming group  was gently groomed and scratched focusing on the most appreciated areas, considered as being positively perceived by horses30. The Standard grooming group  was groomed according to the standard method used in equestrian centres and taught in most horse manuals. This method is a fixed procedure that does not particularly consider the horse’s reactions. From preliminary observations, we hypothesised that these situations would lead to contrasting behavioural responses from horses towards the experimenter: contact-seeking in the first case and avoidance in the second case. We expected differences in facial expressions  according to the subjective experiences of the horses during grooming. We also hypothesised an effect of endocrine responses after the session of grooming, with a higher level of oxytocin and a lower level of cortisol in the Gentle grooming group than in the Standard grooming group. Finally, the measures of heart rate and heart rate variability should provide information regarding the level of arousal associated with each type of grooming. If grooming procedures impact specifically valence and not arousal, we would expect no change in heart rate or heart rate variability.This study aimed to apply two contrasting situations in terms of emotional valence and to determine whether they were associated with specific behaviours, facial expressions and physiological responses . We controlled the type of inductive stimulation by focusing only on somatosensory stimulation: tactile contacts. This type of stimulation  is interesting to study because it can generate opposite emotional responses depending on its characteristics, which can be expressed either by avoidance . Groups differed significantly for all the modes recorded Table  except f1. Groups1,73 = 1.15; P = 0.28). However, an interaction between the group and the session was found . Post hoc analyses revealed that oxytocin levels became significantly lower at S11 in the Gentle group while levels for the Standard group stayed constant , but it did not differ significantly between the groups . The other behaviours were expressed by less than 10% of the animals and were thus not analysed.By contrast, three modalities of the facial expressions continued to differ significantly between groups: Contracted lips ; Upper lip extended and immobile  and White of the eyes . Horses from G group had their lips extended and immobile more frequently, while S horses contracted their lips more frequently, and the white of their eyes was more visible.This study aimed to apply two situations of grooming (Gentle and Standard) and to determine whether they were associated with specific behaviours, facial expressions and physiological responses. We first showed that at the end of the treatment (S11), the two types of grooming induced contrasting behaviours of approach and avoidance indicating opposite emotional valences: positive in the first case and negative in the second. We then identified very different facial expressions according to the group, both at the end of the experiment and one year later when the horses in both groups underwent an identical grooming protocol. This suggests that observing facial expressions is a very sensitive method to assess the emotions felt by an animal during grooming, even when the animals no longer express any particular behaviour. However, physiological results were not as expected: blood cortisol and oxytocin never differed between before and after the grooming session. Interestingly however, after the 11 sessions, basal oxytocin levels were lower in the G group. Finally, heart rate and heart rate variability did not differ between groups.4 . These clear behavioural differences identify two opposite emotional feelings: positive in the first and negative in the second case. These findings are very much in line with previous data which have shown that grooming can induce contact-seeking when preferred zones are massaged44, but when the horse’s reactions are not taken into account it can lead to strong avoidance and defensive reactions (preliminary observations in the field). A limit to this behavioural analysis is that the observer could not be blind to the treatment while recording the behaviours, since he could see how the experimenter was grooming the horse. Even though the behaviours recorded were easy to distinguish, and contrasted greatly between groups, we cannot exclude that this could have introduced a bias. However, this limitation did not exist for the following analyses based on the facial expressions. Indeed, these facial expressions were recorded under a blind condition, as they were based on the pictures of the head of the horses that did not allow the kind of grooming to be seen.We used tactile stimulations to induce contrasting subjective experiences in horses. We hypothesised that an animal feeling a situation positively would seek contact, while an animal feeling a situation negatively would seek to avoid it. In line with what was expected, the horses in the “Gentle grooming” group clearly sought human contact and never expressed discomfort or defensive behaviours. In contrast, the horses from the “Standard grooming” group presented numerous discomfort, defensive and avoidance behaviours. We argue that these behaviours inform us about the animal’s emotional valence, as described in the Core affect theoryThese blind analyses of facial expressions at S11 revealed significant differences between groups during grooming. G and S horses were clearly separated along factor 1 of the PCA. Compared to the Standard grooming horses, the Gentle grooming horses were more often observed with their necks moderately raised, their eyes half closed and their upper lips extended and either immobile or twitching and their ears pointing backwards almost in line with the nose. In contrast, horses from the Standard grooming group were more frequently observed with their neck in a high position, eyes wide open or showing the whites, contracted lips with the corner of the mouth raised jerkily and asymmetrical ears. Moreover, data show that some of these expressions are very specific to one or other of the groups , while other expressions can be observed in the two groups, but at different frequencies . Finally, even though we analysed the facial expressions at S11 only, it is possible that some horses from G group would have expressed the positive facial expression from the first session if they had appreciated the grooming, these expressions could in fact represent an automatic response to a positive emotion as induced by mutual grooming.13. However, certain features of the face have already been identified in a positive context. For example, this is the case of the angle between the line through the eyeball and the highest wrinkle which is reported to decrease during grooming in horses19. Our results supplement these previous observations, and show that other parts of the face, such as the position of the lips or the ears, are also of interest.Taken together, these results suggest that facial expressions could be used to identify subjective experiences in horses in a grooming situation, and particularly positive affects. To date facial expressions in horses have mostly been described in the context of painful or stressful experiences45) and provides evidence that these can be essential in detecting emotions at least in species that have clearly distinguishable characteristic facial expressions.The most striking finding of this study was that one year later, despite handling both groups in a similar manner, the horses again expressed distinctive facial expressions similar to those identified in the first part of the study. These expressions were less contrasted, with fewer modalities differing significantly, but the results were very clear and in line with the observations from the previous year. The upper lip was more frequently extended forwards and immobile in the G group, and contracted with the corner raised jerkily in the S group. Regarding the eyes, they were more often wide open in group S. These results seem even more surprising considering that when analysing the behavioural elements alone nothing distinguished the two groups. The fact that the grooming procedure in year two did not take into account the horses’ reactions probably explains why the difference in behaviours between groups was less prominent than in the first year. However, the results of the facial expressions show that horses had a memory of the relationship established one year previously, despite being groomed with this intermediate procedure. Facial expressions would thus seem to be more sensitive and refined indicators of horses’ feelings than behaviours. This supports different precursory studies on animal facial expressions . For instance, it is fascinating to notice certain similarities between the lip smacking of chimpanzees and extended lips of horses. Moreover, the ear position directed backwards in the G group is interestingly also found in sheep when being stroked40. The fact that several species can show similar expressions, could be explained by their similar evolutionary pattern. For instance, lip movement could stem from mutual grooming itself, which involves mouth movements and which could have become ritualised with this more discrete lip movement. In any case, it would seem reasonable that these facial expressions would have a communicative use at least between conspecifics, but even possibly between species.In future work it would be interesting to compare the facial expressions identified here with those of horses conducting mutual grooming. According to preliminary observations, they would seem to be very similar. This would indicate that horses could have the same facial expressions during interactions with both intra- and inter-specifics, such as with humans. It would be of interest in future studies to test whether horses use these expressions as a means of communication to indicate to a conspecific its intention to engage in an exchange (the case of G-type expressions), or on the contrary, to avoid doing so (type S expressions). For example, in chimpanzees it has been demonstrated that lip smacks play a role in coordinating and prolonging social grooming, a typical example of behaviour facilitating cooperation49. Cortisol level did not decrease after the gentle grooming either, as could have been expected if it was a marker of positive emotions as suggested in some studies . Our finding does not support the hypothesis that tactile stimulation via gentle grooming would elicit an increase in peripheral oxytocin levels. We posited this hypothesis because many studies have reported an oxytocin release in response to tactile stimulation in adult rats50 and humans36 and also during positive social contact including touch when dogs interact with their owner51 or in humans when parents and infants interact52. The lack of consistency with our results may reflect differences in experimental situations or sampling procedures. In particular, the relevance of measuring oxytocin at the plasma level has sometimes been questioned, as brain oxytocin neuropeptide is considered to be a much more reliable candidate53. In lambs which have been able to develop a strong bond with a human, oxytocin plasma levels are found to decrease, rather than increase, when they reunite and interact with their human caregiver after a period of social isolation37. By contrast, within the brain, oxytocinergic neurons are activated in the paraventricular nucleus of the hypothalamus in lambs that are able to interact with their caregiver compared to lambs which remain isolated54. Indeed, when studying oxytocin it is important to choose carefully the method used to investigate it55. It does not mean that peripheral oxytocin is irrelevant and our study shows that plasma levels were affected by our treatment. Indeed, we found an unexpected but interesting result: oxytocin levels became significantly lower at S11 in the Gentle group, both before and after the session. An explanation would be that the basal levels of oxytocin might have been lowered in the G group horses because the repetition of gentle grooming sessions may have induced a general state of well-being in the animals, which was not the case at the start of the handling sessions. On the other hand, horses groomed in a standard manner would not have reached this well-being state because the treatment had maintained a more negative emotion. Such a physiological state is observed in prairie voles that are subjected to chronic social isolation56 or in lambs reared without a mother57. In humans, higher basal levels of oxytocin in plasma have also been associated with greater relational distress60. In addition, it is known that oxytocin has an anxiolytic and anti-stress effect, and can be more elevated in periods of distress, thus down regulating the hypothalamic-pituitary-adrenal axis61. This could also be the case in horses, but further research is required to confirm this result and understand the underlying mechanisms.Regarding the endocrine responses, contrary to what was expected, we did not find any changes after either type of grooming. No rise in cortisol was observed in horses groomed in a rough manner despite their avoidance behaviours and their specific facial expression. It is possible that the situation of standard grooming was not stressful enough for the horses to induce a rise in cortisol, unlike what can be observed in horses after social isolation, a novel environment or stressful husbandry procedures63, there were no statistical differences between groups. In contrast with studies in horses showing that positive stroking could have a calming effect and thus lower heart rate parameters30, there is no conclusive evidence of a relaxing effect due to stroking in the G group. This is in line with the behavioural observations that suggest a relatively high level of arousal in both groups. Indeed, horses presented active behaviours in both groups: S horses showed avoidance behaviour while G horses showed seeking behaviours. Horses also had tense facial expressions in both groups. In fact, G horses did not present relaxed faces like those observed during rest with the lower lip hanging in a relaxed way, but instead they had tense faces with ears pointing backwards and the upper lip extended forwards. Thus, taken together, these results suggest that on the Core affect graph65, the two groups could be distinguished on the emotional valence axis while being relatively similar on the arousal axis.Finally, regarding the heart rate and heart rate variability, considered as markers of emotional arousalTo conclude, this study demonstrated that horses presented contrasting behaviours according to the kind of grooming experienced (Gentle vs Standard), suggesting opposite emotional valences, and that specific facial expressions were associated with them. These expressions appear to be more sensitive than behavioural indicators because they alone enabled different emotions to be detected according to the group one year after the end of treatment. In line with previous studies, this new work confirms that research on facial expressions is a promising avenue in the field of animal emotions. In the present study, the facial expressions identified were probably specific to the context of grooming, and a lot of other facial expressions remain to be identified in different contexts and for different emotional valences and/or levels of arousal. Regarding endocrine responses, despite the unexpected results, the differences in basal oxytocin levels observed between groups at the end of treatment lead the way to further research to confirm whether or not basal oxytocin could also be a marker of well-being in horses. Finally, this study has shown to what extent grooming practices, which could appear to be inoffensive, have a long-term incidence on the horse’s general well-being and the human/horse relationship. This study thus serves as a basis to promote respectful and safe animal welfare practices in the field based on careful observations of behaviours and facial expressions.The protocol was approved by the ethics committee of Val de Loire . Animal care and experimental treatments complied with the guidelines of the French and European guidelines for housing and care of animals used for scientific purposes (European Union Directive 2010/63/EU) and were performed under authorization and supervision of official veterinary services .The study was conducted on 27 Welsh mares, aged 2 (n = 19) or 3 years old (n = 8). From birth until the beginning of the experiment they lived in large groups at pasture during summer and in large inside stalls all together with access to a paddock in winter. They were fed ad libitum with straw and hay. They were checked daily for their general state, but never groomed.From three months before the beginning of the experiment to one week before, all horses were handled twice a week for five minutes to habituate them to a halter, and to being attached in a loose box and touched all over their bodies. Nine days before the beginning of the experiment, horses were tethered for five minutes a day in the testing arena (see below). This initial handling phase was carried out by a person who was not involved in the experiment. The horses were then randomly divided into two groups, balanced for age and weight: Gentle grooming  and Standard grooming . For the two weeks of treatment, the two groups lived together at pasture at night. From 9:00 am until 4:00 pm, they were housed in triads in large loose boxes (5.5 × 3.5 m) combining horses from each group. After the experiment, they returned to pasture with exactly the same husbandry routine as before.Horses were handled by the same female experimenter for eleven 10-min sessions of grooming per day S1 to S11). These sessions were conducted on six consecutive days followed by one day off, and then on five more consecutive days , then with a hard-bristled dandy brush for 2 min 12 and finally with a soft body brush for 1 min 08. Each brush stroke was of medium intensity covering around 30 cm with approximately 80 strokes per minute. This standardized procedure was maintained independently of the horse’s reaction.The experimenter groomed each horse in this group for 10 minutes per session with a procedure which aimed to maximise positive reactions and minimise negative reactions. During each session, grooming began on the left side of the horse and lasted for 4 minutes, and involved using the hands to scratch the horse’s neck, then head, back, belly and hindquarters, and finishing on each leg. When the horse demonstrated approach behaviours , and were no longer groomed. A year later, 10 S horses and 12 G horses were tested again by the same experimenter during a session of 2 min 30.Since the aim was to determine whether the horses had a memory of the grooming sessions conducted one year previously, we choose to carry out a short session (2 min 30 instead of 10 min), to minimise the influence of a new training effect. During this session, horses were tethered with two leading reins in the middle of the testing arena . The grooming procedure was the same for both groups and was intermediate to those used previously in the two treatments. The experimenter gently brushed the different zones of the horse’s body with a plastic currycomb, in the same order as detailed above. No pressure was applied with the brush. The procedure was not adapted according to the horse’s positive or negative reactions, and did not involve scratching preferential zones.th session of grooming and during the single test carried out a year later , directly by the same observer during the 1112, and from three pilot studies which aimed to describe for the different parts of the horse’s head the different modes that are expressed in different contexts . In each case, at least two observers recorded the modes from video footage. One was naïve , while the other was accustomed to observing horses. Only the measures that present good reliability between the observers were selected and reused in the present experiment. Moreover, to check the inter-observer reliability, 20% of the data of the present experiment were reanalysed by a second observer (OBS 2), also blind to the treatment . The level of observers’ agreement on the facial expressions was high .We analysed the positions of the neck, eyelids, lips and ears from the video footages from session 11 and from the single test carried out a year later  were recorded at S11 during the 10-min grooming session . For instance, when the horse’s ears were observed N times in the “asymmetrical ears” position in the 20 images taken during S11, the frequency for the mode “asymmetrical ears” for that session was N/20. Due to the number of animals and the distribution of the data, non-parametric statistics were used to analyse the behavioural data, facial expressions, heart rate and heart rate variability (Mann-Whitney U test for inter-group comparisons). Two-tailed tests were always used. Data are presented as median . The facial expressions (frequency of occurrence of each mode) were also analysed using a Principal Component Analysis (PCA) to give an overview of potential facial expressions specific to each group. In this PCA, the different modes of facial expressions were analysed as active variables and the groups as illustrative variables. Two modes (“Low neck” and “Ears pinned back”) were not included in the PCA because they were expressed by hardly any animals (only 2 horses out of 27 were observed with a low neck or ears pinned back). Due to the distribution of the data, the PCA was based on Spearman correlations. The scores of the subjects of each group on the first two factors of the PCA were also compared with a Mann-Whitney U test. These analyses were performed using Xlstat software  with significance accepted at P ≤ 0.05. We present the original P values because in cases of low sample size, the usefulness of using corrections for multiple comparisons is highly debated and results in a loss of power68.For each horse, the number of times each behaviour was expressed during the 11Oxytocin and cortisol were analysed with linear mixed-effects models (lmm) in R  using the lmer function from the lme4 package. Models were fitted with restricted maximum likelihood estimation (REML) and p-values were derived using the Satterthwaite approximations (anova function from the lmerTest package). These models included oxytocin or cortisol as a response variable, the session number (S1 or S11), the factor "before or after session" and the treatment group (G or S) as fixed factors, as well as all possible interactions between them. The individual identity of the horses was included as a random factor. Non-significant interactions were removed from the final models . Residuals were checked graphically for normal distribution and homoscedasticity. Post hoc analyses were conducted using a Tukey’s test.The datasets generated and analysed during the current study are available from Zenodo (10.5281/zenodo.1321000).Supplementary information


Age-related hearing loss (ARHL) is associated with weakened inhibition in the central auditory nervous system including the cochlear nucleus. One of the main inhibitory neurons of the cochlear nucleus is the D-stellate neuron, which provides extensive glycinergic inhibition within the local neural network. It remains unclear how physiological activities of D-stellate neurons change during ARHL and what are the underlying mechanisms. Using in vitro whole-cell patch clamp technique, we studied the intrinsic membrane properties of D-stellate neurons, the changes of their firing properties, and the underlying mechanisms in CBA/CaJ mice at the ages of 3–4 months (young), 17–19 months (middle age), and 27–33 months (aged). We found that the intrinsic membrane properties of D-stellate neurons were unchanged among these three age groups. However, these neurons showed decreased firing rate with age in response to sustained auditory nerve stimulation. Further investigation showed that auditory nerve-evoked excitatory postsynaptic currents (EPSCs) were significantly reduced in strength with age. These findings suggest that D-stellate neurons receive weakened synaptic inputs from the auditory nerve and decreased sound driven activity with age, which are expected to reduce the overall inhibition and enhance the central gain in the cochlear nucleus during ARHL. The cochlear nucleus (CN) is the first neural station in the central auditory pathway that processes all sound information from the periphery auditory nerve and projects to higher auditory nuclei . It is wOne of the major CN inhibitory interneurons is the D-stellate neuron in the ventral cochlear nucleus (VCN) ,16,17,18In this study, we investigated the physiological changes of the D-stellate neurons in VCN during ARHL in three age groups of CBA/CaJ mice at 3–4 months , 17–19 months (middle age with mild hearing loss), and 27–33 months (aged with ARHL). We found that the D-stellate neurons had similar intrinsic membrane properties among all three age groups of mice, suggesting that these inhibitory interneurons are intrinsically normal in function during aging. However, D-stellate neurons showed decreased firing rate with age in response to long trains of auditory nerve stimulation. Voltage clamp experiments further showed that the synaptic drive from the auditory nerve to D-stellate neurons was significantly weakened with age. The findings suggest that sound driven activity in D-stellate neurons is decreased during aging, which contributes to the weakened inhibition in the CN. The decreased firing rate in D-stellate neurons is the result of weakened synaptic drive from the auditory nerve during ARHL. All experiments were performed under the guidelines of the protocols approved by the Institutional Animal Care and Use Committee at The Ohio State University.n = 19), 17–19 months , and 27–33 months . Mice were initially purchased from the Jackson Laboratory, bred and maintained at the university animal facility until the age of experimental use.CBA/CaJ mice of either sex were used at three age groups: 3–4 months  or a VT1200S Microtome  ,33. The 3-based electrode solution that contained (in mM): 130 CsMetSO3, 5 CsCl, 5 EGTA, 10 HEPES, 4MgATP, 0.3 GTP, 10 Tris-phosphocreatine, and 3 QX-314. To activate the auditory nerve inputs, electric pulse stimulation was delivered through a 75-µm diameter concentric stimulating electrode , which was placed at the area of the auditory nerve root. Two µM strychnine was added to the ACSF bath to block glycinergic inhibitory transmission in all recordings. Membrane potentials from current clamp recordings were adjusted with a junction potential of −12 mV. Cochlear nucleus slice was transferred to the recording chamber under Axio Examiner microscope , submerged in ACSF bath with a constant flow of 2–3 mL/min and the temperature controlled at 34 °C throughout the recording session. Recording pipettes were pulled from borosilicate glass  with a Sutter P2000 puller . Data acquisition was made using a Multiclamp 700 B amplifier, Digidata 1550 B acquisition system, and pClamp 11 software (Molecular Devices). Whole-cell patch clamp recording was performed under either current clamp or voltage clamp mode. For current clamp recordings, glass pipettes were filled with K-gluconate-based electrode solution that contained (in mM): 126 K gluconate, 6 KCl, 2 NaCl, 10 HEPES, 0.2 EGTA, 4 MgATP, 0.3 GTP, and 10 Tris-phosphocreatine, with pH adjusted to 7.20. For voltage clamp recordings, glass pipettes were filled with Cs-MetSOAll electrophysiological recordings were made from the anteroventral cochlear nucleus (AVCN), where three types of neurons were encountered in our experiments including bushy, T-stellate, and D-stellate neurons. Only D-stellate neurons were included in this study based on their distinctive features in morphology and electrophysiology . Alexa FData were analyzed using Igor Pro software  with custom-written functions. Intrinsic membrane properties were analyzed from all D-stellate neurons with current clamp data, as described in previous studies ,32. The GraphPad Prism  was used in all statistical analyses. Data were presented as mean ± standard deviation.n = 19), which was significantly lower than the thresholds of the middle age  and the aged mice  . Hearing thresholds could not be determined from five aged mice because no clear ABR waveforms were evoked by clicks at even the highest tested level of 90 dB SPL. These five mice were not included in In order to test the hearing status of the CBA/CaJ mice in this study, we measured the ABRs from all mice in three age groups to clicks at sound levels from 20 to 90 dB SPL. Hearing threshold was determined as the minimum sound level that evoked identifiable ABR waveforms as shown in p = 0.690). The average membrane input resistances were 108 ± 54 MΩ (young), 102 ± 42 MΩ (middle age), and 113 ± 33 MΩ (aged) (p = 0.473). The membrane time constants were 5.3 ± 2.4 ms (young), 4.6 ± 1.4 ms (middle age), and 4.6 ± 1.6 ms (aged) (p = 0.857). The threshold currents, which were the minimum current injection level that evoked action potentials, were 75 ± 35 pA (young), 100 ± 88 pA (middle age), and 112 ± 54 pA (aged) (p = 0.289). The magnitude of depolarization sag in response to hyperpolarizing current injection was quantified as the ratio of hyperpolarization levels at the steady state versus the peak (Ih b/a ratio). In three groups mice, the ratios were 0.57 ± 0.09 (young), 0.56 ± 0.07 (middle age), and 0.50 ± 0.07 (aged), respectively (p = 0.088). The decay time constants of the depolarization sags were 35 ± 15 ms (young), 33 ± 14 ms (middle age), and 21 ± 7 ms (aged) (p = 0.055). The average spike amplitudes were 64 ± 7 mV (young), 65 ± 9 mV (middle age), and 61 ± 7 mV (aged) (p = 0.433). The average spike half-widths were 0.34 ± 0.06 ms (young), 0.36 ± 0.11 ms (middle age), and 0.40 ± 0.11 ms (aged) (p = 0.556). The average spike thresholds were −39.4 ± 5.2 mV (young), −36.7 ± 3.7 mV (middle age), and −39.4 ± 3.9 mV (aged) (p = 0.319). In summary, D-stellate neurons from three age groups of mice showed no change in their intrinsic membrane properties, which suggests that these neurons themselves are functionally stable during aging. To evaluate the intrinsic membrane properties of D-stellate neurons, we performed whole-cell patch clamp recording under current clamp mode using acute brain slices from mice at all three age groups. D-stellate neurons were identified based on their morphological and electrophysiological features as described in Materials and Methods. Data were collected from 9 D-stellate neurons from the young mice, 6 from the middle age mice, and 13 from the aged mice. Intrinsic membrane properties of all neurons were measured from current step injection responses (p < 0.0001) and age (p < 0.0001), with no interaction effect between the two (p > 0.9999). Similar changes in firing patterns with age were also observed in D-stellate responses to 400 Hz stimulus trains . The actual firing rates to 100 Hz stimulus trains were 0.86 ± 0.45 spikes/stimulus in young mice, 0.40 ± 0.25 spikes/stimulus in middle age mice, and 0.26 ± 0.26 spikes/stimulus in aged mice (p = 0.008); and the firing rates to 400 Hz stimulus trains were 0.56 ± 0.36 (young), 0.29 ± 0.16 (middle age), and 0.13 ± 0.11 spikes/stimulus (aged), respectively (p = 0.014).It is well-known that the auditory system is highly active, with auditory nerve fibers that can sustain firing at rates up to hundreds of Hz ,40. As os trains B and sump = 0.964). The spike temporal precision was also similar in D-stellate neurons at 400 Hz among different age groups, with the vector strength of 0.13 ± 0.08 (young), 0.14 ± 0.10 (middle age), and 0.17 ± 0.14 (aged), respectively  but not at high rates (200–400Hz) . To testThe findings that D-stellate neurons do not change in their intrinsic membrane properties during aging, but decrease in firing rate to long trains of auditory nerve inputs suggest that the excitatory synaptic drive is weakened with age in D-stellate neurons.p = 0.182), which suggests that the synaptic strength of the auditory nerve terminals onto D-stellate neurons do not change with age under quiescence. We also recorded the paired pulse stimulation evoked eEPSCs, and found no significant difference in paired pulse ratio at either 10-ms pulse interval (p = 0.549) or 2.5-ms pulse interval  from D-stellate neurons under voltage clamp mode in response to single auditory nerve stimulation A. The avp < 0.0001) and with stimulus number (p < 0.0001), with no interaction between the two (p > 0.9999). Similar differences were found in eEPSC trains at 400 Hz . Given that the auditory system is highly active under physiological condition and D-stellate neurons decrease in firing with age at high rate activities , we furtThe results showed that during aging, D-stellate neurons receive weakened sensory inputs from the auditory nerve with reduced synaptic drive, and fire less spikes during sustained activities. The decreased firing in D-stellate neurons would reduce the overall inhibition within the neural network and promote the response of other CN neurons to auditory nerve inputs. One of the prevailing theories in brain aging is that inhibition diminishes with age, which leads to compromised neural function, including in the auditory system . In CN, The finding that D-stellate neurons show no change in intrinsic membrane properties among three age groups of mice was unexpected. In contrast, the excitatory bushy neurons in aged mice have depolarized membrane potential, increased membrane input resistance, and decreased threshold current, which lead to enhanced excitability that would counteract the reduced sensory inputs during ARHL . SimilarDecreased sensory inputs from the auditory periphery during hearing loss is known to be compensated by enhanced gain in the central nervous system ,44. DespTo date, much more focus of ARHL has been on changes in the periphery auditory system ,46,47,48This study investigated the physiological changes of the inhibitory D-stellate neurons of the VCN in three age groups of mice. We found that D-stellate neurons maintain stable membrane properties, but decrease in neuronal activity during aging because of weakened synaptic drive from the auditory nerve inputs. We conclude that the reduced activity in aged D-stellate neurons decreases the overall inhibition and enhances the central gain in the CN, which serves as a compensatory mechanism to counteract the reduced sensory inputs from the auditory periphery during ARHL.


Many pairwise meta-analyses (MAs) related to therapies of varicose veins have been published, but their reporting and methodological quality remain unclear. The present study was designed to assess the overall quality of pairwise MAs related to therapies of varicose veins.We will systematically search 4 electronic databases, including PubMed, EMBASE, Cochrane Library, Chinese Biomedical Database, to identify pairwise MAs related to therapies of varicose veins. The search time-span was set from inception to March 2019. The pairwise MAs related to therapies of varicose veins will be included in our overview. The reporting and methodological quality of included MAs will be assessed using preferred reporting items for systematic review and meta-analysis and a measurement tool to assess systematic reviews 2, respectively. Meanwhile, we will extract some general characteristics of included MAs, including first author; published year, journal, sample size, number of studies, number of randomized controlled trials and intervention details, and so on. All literatures screening, quality assessment, and data extraction will be independently completed by 2 of all reviewers, and any disagreement will be resolved by discussion. Besides, an increasingly popular method – evidence mapping, will be used to present the whole evidence landscape related to therapies of varicose veins. The assessment results will be presented as percentage and event/total. The Excel 2016 will be used to manage and analyze data.The results of the overview will be submitted to a peer-reviewed journal for publication.This overview will summarize the overall reporting and methodological quality related to therapies of varicose veins.CRD42019126722. Although some patients with varicose veins remain asymptomatic, others may experience pain, aching, heaviness, and itching, that can seriously affect quality of life. It also poses big financial burden; a recent report shows that approximately 2% of national healthcare resources was spent on treatment of varicose veins in London. In practice, varicose vein treatments including conservative therapy, surgery involving saphenous ligation and stripping, and others new treatments.,6,7,8 Until recently, surgery was considered as standard treatment, it also with high ligation and stripping to knee level, and combing with phlebectomies.,10,11 However, operation may occasionally be associated with significant postoperative morbidity, including bleeding, thrombophlebitis and groin infection, and so on.Varicose vein is one of the most commonly chronic venous diseases, and prevalence for varicose veins is higher, 1% to 73% in females and 2% to 56% in males in Western countries.,13,14 related to therapies of varicose veins have been published, but their reporting and methodological quality remain unclear. However, the reporting and methodological quality of MAs very important for clinicians and clinical practice guideline developers to judge their reliability. Therefore, the guideline – preferred reporting items for systematic reviews and meta-analyses (PRISMA), which was developed and as a checklist was recommended to report MAs. And a well-known tool – a measurement tool to assess systematic reviews (AMSTAR), which was developed and recommended to assess the methodological quality of MAs or as a methodology guideline for conducting of MAs, and it was upgraded to AMSTAR-2 in 2017.The meta-analyses (MAs) is a review that always using systematic approaches to identify, select and critically appraise primary studies. And many pairwise MAs,19 will be used to present the whole evidence landscape.To the best of our knowledge, pairwise MAs related to therapies of varicose veins has not been apprised using PRISMA and AMSTAR-2. Therefore, the present study was designed to assess the overall quality of pairwise MAs related to therapies of varicose veins, and a recent popular method – evidence mapping (EM),2 and the reporting of this study results will follow PRISMA guideline. This overview has been registered on the international prospective register of systematic reviews.This protocol follows PRISMA-P,2.1We will include pairwise MAs related to therapies of varicose veins, and they should be published in Chinese or English. Some publication document types will not be considered, such as letter, comment, and conference abstract, and so on. If there are duplicated papers for 1 special research, we will include the latest one and could provide the most information on quality of the research.2.2We plan to search the following databases to identify pairwise MAs related to therapies of varicose veins from inception to March 2019: PubMed, EMBASE, The Cochrane Library, Chinese Biomedical Database. Language will be restricted as English or Chinese. The search strategy of PubMed was as:#1 “Varicose Veins” [Mesh]#2 “chronic lower extremity venous insufficiency” [Title/Abstract]) OR “varicose veins” [Title/Abstract]) OR “varicose vein” [Title/Abstract]) OR “veins varicose” [Title/Abstract]) OR “vein varicose” [Title/Abstract]) OR Varix∗ [Title/Abstract]) OR Varice∗ [Title/Abstract]) OR varicosi∗ [Title/Abstract]) OR “enlarged twisted veins” [Title/Abstract]) OR “tortuous veins” [Title/Abstract]) OR “saphenous vein” [Title/Abstract]) OR “saphenous veins” [Title/Abstract]) OR “varicose venous” [Title/Abstract]#3 #1 OR #2#4 “Systematic Reviews as Topic” [Mesh] OR “Systematic Review” [Publication Type] OR “Meta-Analysis as Topic” [Mesh] OR “Meta-Analysis” [Publication Type]#5“systematic review” [Title/Abstract] OR “systematic reviews” [Title/Abstract] OR “meta analysis” [Title/Abstract] OR “meta analyses” [Title/Abstract] OR metanalysis [Title/Abstract] OR metanalyses [Title/Abstract] OR meta-analysis [Title/Abstract] OR meta-analyses [Title/Abstract]#6 #4 OR #5#7 #3 AND #62.3Initial records from electronic databases will be imported into the EndNote X8 software. First, the titles and abstracts of all records will be reviewed independently by 2 reviewers. Then, full text of all potentially relevant MAs will be retrieved to make the final decision. Any conflict will be resolved by discussion. A flow diagram will be used to describe the process of MAs selection Fig. .2.42.4.1 The developers of the PRISMA proposed 3 answer options for each item: yes, no and partial. We will use PRISMA checklist to assess each item of the included MAs. Finally, we will count the percentage and event of MAs who answer “yes,” who answer “partial” and who answer “no” for each item. The assessed result will be presented according to the form of Table The PRISMA checklist including 7 parts with 27 items.2.4.2 which consists of 16 items. In our study, we will take AMSTAR-2 to assess each included MA and count the percentage and event who answer “yes,” “partial yes” and “no” for each item. The overall confidence on the results of the MAs will be rated according to the following 4 categories: High , Moderate , Low , Critically low . Finally, we will count the percentage and event of each items, and categories of the overall confidence on the included MAs ; the rating of authors’ conclusions  of the MAs (x-axis), the number of primary studies included in each MA (bubble size), and a pie will be used to show the proportion of randomized controlled trials.We will use EM method to present the evidence landscape related to therapies of varicose veins. The presentation form of EM will be using the bubble plot and, which will display information on 4 dimensions: AMSTAR-2 assessment  form will be used to extract and analyze data from the included MAs. Extracted information will include: first author; published year; journal; funding; sample size; number of studies; number of studies; details of interventions and control conditions; effect size and confidence interval; outcomes; conclusions and contents related to PRISMA and AMSATR-2. And the assessment results and other data will be presented as percentage or (and) event/total.2.7Because this study is not a clinical study, and we will search and evaluate only existing sources of literature. So ethical approval is not required.3To the best of our knowledge, this is the first overview to assess overall quality of pairwise MAs related to therapies of varicose veins by using PRISMA and AMSATR-2. Meanwhile, a popular method-EM, will be used to present evidence. And we believe the results of this overview will provide some references for clinicians and clinical practice guideline developers.Jie Ding, XiaoFei Mu, and Cai Liu conceived the study. XiYun Zhao and LiNa Qing designed the flowchart of literature selection and 2 tables. Yuan Yuan and KongXi Wei designed the search strategy. Yuan Yuan and LiYao Tang participated critical revision of the manuscript. All the authors contributed to draft and approve the study protocol. Jie Ding will supervise the overall conduct of the study.Conceptualization: Jie Ding, XiaoFei Mu, Cai Liu.Investigation: Yuan Yuan, LiYao Tang, KongXi Wei.Methodology: XiYun Zhao, LiNa Qing.Resources: Yuan Yuan.Writing – original draft: Jie Ding.Writing – review and editing: Jie Ding, Yuan Yuan, Cai Liu.


This questionnaire was initially adapted into Spanish (S-MARS-HA) using forward and backward translations by bilingual English-Spanish speakers. A sample of 252 older adults fitted with hearing aids at a public hospital in Santiago, Chile, was investigated. Educational level was measured as the number of years of formal education. Participants responded to the S-MARS-HA along with questions exploring social support, attitudes in using hearing aids, participation in social events, and vision and joint problems. Hearing aid adherence was investigated with the use of a question from the International Outcome Inventory for Hearing Aids. All these procedures were conducted at the participants’ homes. Pure-tone average  in the fitted ear was obtained from the participants’ medical records. Univariate and multivariate regression models were constructed to investigate the association between educational level and hearing aid self-efficacy controlling for the covariates of interest . The S-MARS-HA showed an adequate construct validity along with a good reliability. Results of the multivariate regression analyses showed that educational level significantly predicted hearing aid self-efficacy. Covariates significantly associated with this outcome included attitudes in using hearing aids and PTA in the fitted ear. Finally, a significant association between hearing aid self-efficacy and adherence to hearing aid use was observed. In conclusion, this study showed a significant association between educational level and hearing aid self-efficacy in older adults from a developing Latin American country. Thus, this variable should be considered when designing and delivering aural rehabilitation programs such as hearing aids to older adults, especially those from developing countries.Hearing aids are the most common rehabilitation strategy for age-related hearing loss. However, 25% to 50% of older adults fitted with hearing aids do not wear them post-fitting. Hearing aid self-efficacy has been suggested as one of the key factors that may explain adherence to hearing aids in older adults. The primary aim of this study was to determine a possible association between educational level and hearing aid self-efficacy in older adult hearing aid users from a Latin American country . The secondary aim was to determine if in this sample of older adults, hearing aid self-efficacy predicted hearing aid adherence as previously suggested by other studies. The MARS-HA  Hearing aids are the most common rehabilitation strategy for age-related hearing loss. However, 25% to 50% of older adults fitted with hearing aids do not wear them post-fitting –3. A numHearing aid self-efficacy has been defined by Smith and West  as the iTherefore, understanding the variables that can predict hearing aid self-efficacy in older adults seems to be crucial for successful hearing aid fitting in the clinical setting. Smith and West  found th“enactive mastery experiences” and “verbal persuasion” sources of information. One strategy to increase the “Enactive Mastery Experience” is to divide new skills into smaller steps Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Tittle:Does Educational Level Predict Hearing Aid Self-Efficacy in Experienced Older Adult Hearing Aid Users from Latin America?Version: August 30th, 2019.PLOS ONEReviewer: Fernando GomezThis is a cross sectional observational study in a convenience sample from an urban hearing impairment outpatient program of Governmental hospital in Santiago (Chile). This paper has three components, one is the validity of a questionnaire used to measure hearing aid self-efficacy: the Spanish version of Measure of Audiologic Rehabilitation Self-Efficacy for Hearing Aids (S- MARS-HA); another second component about the role of educational level and other factors as attitudes in using hearing aids and the pure-tone average, (PTA) in the fitted ear as important factor of hearing aid self efficacy and, finally the third component of this manuscript explores the association between hearing aid self-efficacy with adherence to hearing aid use. The topic of the manuscript is appropriate for the Journal. It could be of interest to investigators and clinicians. Minor essential revisions are necessary.Tittle:The title only described one component of the paper, educational level and hearing self-efficacy; any alternative to wide all topics included in the manuscript? However, the title is consistent with the presented problem and reflects the main message of the study.Abstract:Abstract: Concise and specific.Main objective of the study is presented.The statistical methods used to prove the hypothesis is mentioned.Conclusion highlights the contribution of this work.Introduction:What is the rationale of relationship between educational level and hearing aid self-efficacy? Please provide in introduction section short information about it. Only comments about suitability of hearing aid user guides for older people is founded. If not is possible at least the relationship between education and wear hearing aids.About educational level and hearing aid adoption, please check:Popelka M.M., Cruickshanks K.J., Wiley T.L., Tweed T.S., Klein B.E. et al. 1998. Low prevalence of hearing aid use among older adults with hearing loss: The Epidemiology of Hearing Loss Study. J Am Geriatr Soc , 46, 1075 – 1078Tomita M., Mann W.C. & Welch T.R. 2001. Use of assistive devices to address hearing impairment by older persons with disabilities. Int J Rehabil Res , 24, 279 – 289.Kochkin S. 2009. MarkeTrak VIII: 25-year trends in the hearing health market. The Hearing Review , 16, 12 – 31.Material and methods:Sufficient details about the process are provided.Statistical analyses used are appropriate.The methods are appropriate and well described.Results:Information is clearly provided.One figure and six tables are clear and well designed.Supporting information in annex including Spanish version of tool is provided.Discussion:Discussion should no include results, for example about validity and reliability of the S-MARS-HA. Please revise first paragraph of discussion section.Please check second paragraph about comparison of reliability of S-MARS-HA with literature, in this case with Canadian-French version of Stigma Consciousness Questionnaire (SCQ), it is necessary provide more information, rather than similar age in both studies.What is the reference supporting the explanation of weak correlations in several subscales of the tool?To support their findings authors should insist in relationship with self-efficacy and education in previous original paper Bandura`s self-efficacy thesis, see references of Meyer C, Hickson L, Fletcher A. Identifying the barriers and facilitators to optimal hearing aid self-efficacy. Int J Audiol. 2014; 53(suppl 1): S28-S37, and internal locus of control and educational level.Why authors shown results in discussion section?, see “wear of hearing aid”, page 29-30. Time of wearing device is an excellent hypothesis for positive attitude, please reinforce it with appropriate reference. Please check in this reference provided by authors: Ng JH, Loke AY. Determinants of hearing-aid adoption and use among the elderly: a systematic review. Int J Audiol. 2015; 54(5): 291-300.In the same way, paragraph about PTA and participation in social activities as a factor for better adherence to hearing aid deserve other reference.References than support several comments are necessary, for example page 29 paragraph about “ handling hearing aid and educational level or page 29 and 30 about attitudes to hearing aids, please see excellent references provided about barriers to help-seeking for hearing aid adoption.References:There were 32 and all are appropriate.Thanks for letting me review this manuscript.This could be a nice paper.Level of interest: An article whose findings are important to those with closely related research interests.Quality of written English: Well.Statistical review: No.Declaration of competing interests:I declare that I have no competing interest.Reviewer #2: The answer of "no" to Question 4 is only referring to a few minor grammatical issues flagged in the attachment. This is a well-written paper overall, but according to the review instructions I would choose "yes" only if I had no revisions at all.The answer of "no" to Question 3: The instructions state that the data should be included as a supplemental file or publicly available. I was not able to find this in the supplemental file that was included with this submission, nor could I find a website that makes the data available - my apologies if I missed it. I do see your statement that the data will be made available - I will leave it to the editor if this can simply be added as a revision to this paper.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentPlos One S-MARS-HA.docxSubmitted filename: Click here for additional data file. 3 Nov 2019The following changes have been made according to the specific comments previously received:Reviewer #1: This is a cross sectional observational study in a convenience sample from an urban hearing impairment outpatient program of Governmental hospital in Santiago (Chile). This paper has three components, one is the validity of a questionnaire used to measure hearing aid self-efficacy: the Spanish version of Measure of Audiologic Rehabilitation Self-Efficacy for Hearing Aids (S- MARS-HA); another second component about the role of educational level and other factors as attitudes in using hearing aids and the pure-tone average, (PTA) in the fitted ear as important factor of hearing aid self efficacy and, finally the third component of this manuscript explores the association between hearing aid self-efficacy with adherence to hearing aid use. The topic of the manuscript is appropriate for the Journal. It could be of interest to investigators and clinicians. Minor essential revisions are necessary.Tittle:The title only described one component of the paper, educational level and hearing self-efficacy; any alternative to wide all topics included in the manuscript? However, the title is consistent with the presented problem and reflects the main message of the study.R. According a suggestion from the second reviewer (see below) the title was modified with the aim of including the validation process of the questionnaire: Does Educational Level Predict Hearing Aid Self-Efficacy in Experienced Older Adult Hearing Aid Users from Latin America? Validation process of the Spanish version of the MARS-HA questionnaire.Abstract:Abstract: Concise and specific.Main objective of the study is presented.The statistical methods used to prove the hypothesis is mentioned.Conclusion highlights the contribution of this work.Introduction:What is the rationale of relationship between educational level and hearing aid self-efficacy? Please provide in introduction section short information about it. Only comments about suitability of hearing aid user guides for older people is founded. If not is possible at least the relationship between education and wear hearing aids.R. We strongly believe that the effect of educational level on the hearing aid self-efficacy is mediated mainly by suitability of hearing aid user guides for older people. As far as we know, this could be the only variable taken into account by other previous studies. However, as suggested by the reviewer, we have further elaborated this hypothesis. Please see page 4, lines 23-24; page 5, lines 1-14, and lines 18-22 of the revised version of the manuscript. Material and methods:Sufficient details about the process are provided.Statistical analyses used are appropriate.The methods are appropriate and well described.Results:Information is clearly provided.One figure and six tables are clear and well designed.Supporting information in annex including Spanish version of tool is provided.Discussion:Discussion should no include results, for example about validity and reliability of the S-MARS-HA. Please revise first paragraph of discussion section.R. As suggested by the reviewer, we excluded from this paragraph the information about actual results. However, some information about the construct validity was not removed in order to support the explanation about the error variances estimation. Error variances estimation refers to the relationship between items that are likely explained by their association with a variable other than the factor they loaded. According to Byrne (2013) such covariance between items can result from overlapping item content. Therefore, the information about the overlapping item content was kept .References- Byrne BM. Structural equation modeling with Mplus: Basic concepts, applications, and programming. Routledge; 2013.Please check second paragraph about comparison of reliability of S-MARS-HA with literature, in this case with Canadian-French version of Stigma Consciousness Questionnaire (SCQ), it is necessary provide more information, rather than similar age in both studies.R. As suggested by the reviewer, we have now extended this discussion. See page 28, lines 20-23 and page 29, lines 1-2).What is the reference supporting the explanation of weak correlations in several subscales of the tool?R. In the current study, the aided listening sub-scale concentrates the weak correlations. This is similar to the previous validation of MARS-HA questionnaire by West & Smith (2007) where the correlation between Aided listening and Advanced handling sub-scales was weak (r=0.29). In addition, the correlation between Aided listening and basic handling sub-scales was moderate (r=0.46). Although West & Smith did not provide an explanation about these findings, we believe that these correlations may be explained by the fact that the aided listening subscale had relatively lower scores than the other subscales. This could be related to the fact that very few older adults increased their participation in social activities after being fitted with the hearing aid (only 2.4% of the sample), exposing themselves poorly to communicate with others . Another explanation could be that MARS-HA questionnaire includes four different constructs in which different skills are involved. In the case of the Basic and Advanced handling motor skills are involved. On the contrary, for the Aided listening and Adjustment subscales underlie communicative skills. We added more information for explaining the weak correlations in the discussion section .Reference- West RL, Smith SL. Development of a hearing aid self-efficacy questionnaire. Int J Audiol. 2007; 46(12): 759-71.To support their findings authors should insist in relationship with self-efficacy and education in previous original paper Bandura`s self-efficacy thesis, see references of Meyer C, Hickson L, Fletcher A. Identifying the barriers and facilitators to optimal hearing aid self-efficacy. Int J Audiol. 2014; 53(suppl 1): S28-S37, and internal locus of control and educational level.R. Meyer et al. (2014) cited Smith & West (2006), an article where they mentioned the sources of self-efficacy beliefs proposed by Bandura . According to Bandura, individuals formulate self-efficacy beliefs primarily from four sources of information: (a) enactive mastery experiences, (b) vicarious experience, (c) verbal persuasion, and (d) physiological and affective states. Smith & West (2006) provided examples related to audiological rehabilitation for adults with hearing loss in which it is possible to identify the effect of educational level on some of the sources of self-efficacy beliefs previously mentioned. It is possible that the effect of educational level on self-efficacy mainly occurs through “Mastery Experience” and “Verbal persuasion” sources of information. One strategy to increase the “Mastery Experience” is to divide new skills into smaller steps as it is incorporated in the hearing aid user guides for older people, whereas “Verbal persuasion” can be increased by giving appropriate feedback or providing pedagogic materials, such as are provided in clinical controls. In both cases, having a higher educational level would facilitate the aforementioned strategies to increase “Mastery Experience” and “Verbal persuasion” sources of information. We added more information at this respect in the introduction section .References− Bandura A. Self-efficacy: Toward a unifying theory of behavior change. Psychological Review. 1987; 84: 191–192.− Bandura A. Social foundations of thought and action: A social cognitive theory. Englewood Cliffs, NJ: Prentice-Hall. 1986.− Bandura A. Self efficacy: The exercise of control. New York: Freeman. 1997.− Smith SL, West RL. The application of self-efficacy principles to audiologic rehabilitation: a tutorial. Am J Audiol. 2006; 15(1): 46-56. Why authors shown results in discussion section?R. The odds ratio reported was eliminated from this current version of the manuscript.See “wear of hearing aid”, page 29-30. Time of wearing device is an excellent hypothesis for positive attitude, please reinforce it with appropriate reference. Please check in this reference provided by authors: Ng JH, Loke AY. Determinants of hearing-aid adoption and use among the elderly: a systematic review. Int J Audiol. 2015; 54(5): 291-300.R. References were added according to the reviewer’s comment. The studies carried out by Meyer et al. and Hickson et al. included information about the relationship between attitudes, hearing aids self-efficacy and adherence. Meyer et al. found that the length of hearing aid use was a significant predictor of advanced handling subscale. Similarly, Hickson et al. observed that older adults who possessed both a positive attitude towards hearing aids and greater confidence in their ability to manage the more advanced features of a hearing aid were more likely to report a successful outcome with hearing aids .In the same way, paragraph about PTA and participation in social activities as a factor for better adherence to hearing aid deserve other reference.R. Results from studies carried out by Heffernan et al. (2016), and Wu & Bentler (2012) were incorporated. In Heffernan et al. (2016), UK older adults with mild-moderate hearing loss reported activity limitations and participation restrictions (i.e. conversing with the others). Wu & Bentler (2012) recruited North American adults with bilateral hearing impairment aged 40–88 years. Older adults (> 65 years) had less active social lifestyles  requiring fewer demands on hearing than younger adults (< 65 years), matched by hearing thresholds. These studies support our statement that participating in more social activities allows people to experience a richer variety of listening environments, thus providing a sort of listening training with the hearing aid .References- Heffernan E, Coulson NS, Henshaw H, Barry JG, Ferguson M. Understanding the psychosocial experiences of adults with mild-moderate hearing loss: An application of Leventhal's self-regulatory model. Int J Audiol. 2016; 55 Suppl 3: S3-S12. - Wu YH, Bentler RA. Do older adults have social lifestyles that place fewer demands on hearing? J Am Acad Audiol. 2012; 23(9): 697-711.References than support several comments are necessary, for example page 29 paragraph about “handling hearing aid and educational level or page 29 and 30 about attitudes to hearing aids, please see excellent references provided about barriers to help-seeking for hearing aid adoption.R. References were provided accordingly.References:There were 32 and all are appropriate.Thanks for letting me review this manuscript.This could be a nice paper.Level of interest: An article whose findings are important to those with closely related research interests.Quality of written English: Well.Statistical review: No.Declaration of competing interests:I declare that I have no competing interest.Reviewer #2: The answer of "no" to Question 4 is only referring to a few minor grammatical issues flagged in the attachment. This is a well-written paper overall, but according to the review instructions I would choose "yes" only if I had no revisions at all.The answer of "no" to Question 3: The instructions state that the data should be included as a supplemental file or publicly available. I was not able to find this in the supplemental file that was included with this submission, nor could I find a website that makes the data available - my apologies if I missed it. I do see your statement that the data will be made available - I will leave it to the editor if this can simply be added as a revision to this paper.R. In this current version of the manuscript the dataset was uploaded as Supporting Information (S1. Dataset).Overall, the aim of this paper was to investigate whether hearing aid self-efficacy is related to educational level, selecting a population from a Latin American country on the argument that this population may vary in educational level (which turned out to be true in this sample). The self-efficacy measure is published, well-constructed, and has been applied successfully in several past hearing aid studies on a range of topics. The authors applied a rigorous, well-described translation process to this questionnaire in order to develop a Spanish-language version aiming to maintain the questionnaire’s items and scoring methods. The authors also piloted this translation in a sample of 48 participants to ensure that it was feasible for use, prior to applying it in the current study in a well-justified larger sample. In this sample, the authors performed data collection and analysis to answer the primary aim of the study, relating hearing aid self-efficacy to educational level, among several other factors. Overall, this method is reasonable and the question is of interest in our understanding of factors that relate to hearing aid self-efficacy, which on its own has been shown to impact rehabilitation outcomes. It is not surprising that the authors found a relationship between hearing aid self-efficacy and educational level.This is a well-conducted study that makes a good contribution to the literature, and may provide guidance to those who provide hearing rehabilitation services or those who develop hearing aid user’s guides for a wide range of populations.My only concern with this paper is its actual breadth versus its title. With a full translation, feasibility evaluation, and confirmatory factor analysis to not only develop but also to validate the Spanish version of the MARS-HA, this paper has not one but two primary aims. Only the latter is identified in the title. R. The title was modified with the aim of including the validation process of the Spanish version of the MARS-HA questionnaire:Does Educational Level Predict Hearing Aid Self-Efficacy in Experienced Older Adult Hearing Aid Users from Latin America? Validation process of the Spanish version of the MARS-HA questionnaire.Minor comments:Page 6, 2nd paragraph: I don’t think the translated version can be considered “preliminary” and “final” at the same time.R. The word “final” was not really what we meant. We just wanted to mention “preliminary version”. Therefore, the word “final” was deleted from this statement .“Equivalencies” or “equivalence” perhaps rather than “equivalences”R. The word “equivalences” was replaced by “equivalence” .Page 9, bottom:“hours” rather than “hour”R. The word “hour” was replaced by “hours” . Page 11, adherence to hearing aidsYou have chosen to use a self-report measure of adherence rather than the data log of hearing aid use provided by the hearing aid. Please justify this choice (perhaps reading data logs was not feasible in the context of home visits) and include literature  on the validity of self-report. Given the large reporting ranges used on the IOI, minor differences between data logs and self report may not have impacted your analyses, but it would be helpful to the reader to acknowledge any limitations of self-report use metrics, either here or in the Discussion.R. Hearing aid data-logging could not be accessed in this study as data collection was carried out at participants’ homes. We have now provided this explanation in the revised version of the manuscript, including a reference about how accurate self-report metrics can be in this field of research References- Laplante-Lévesque A, Nielsen C, Jensen LD, Naylor G. Patterns of hearing aid usage predict hearing aid use amount (data logged and self-reported) and overreport. J Am Acad Audiol. 2014; 25(2): 187-98.Page 27The sentence starting with “The fact that these pairs of items” needs some editing. I think “other than the factor with which they loaded” might be what you meant.R. The sentence “other than the factor they loaded” was replaced by “other than the factor with which they loaded” .Participants were fitted with hearing aids that did not offer noise management. Why was this unusual fitting profile selected? Is this due to the aids that were available (unlikely) or was it a decision to not provide this? Do you think this explains your low scores on aided listening?R. In Chile, adults aged 65 and older who require hearing aids are provided with one device  either for free or with a maximum co-payment of 20% of the device price (Government of Chile). The latter is determined based on the person’s income levels. Under this programme, hearing aids with multiple channels and up to four programmes whose cost is low (approximately $105 USD) are provided by public hospitals. Each hospital puts out a tender for the contract to purchase the hearing aids and the company chosen fits them and perform follow-up appointments with the patient (this involves three sessions to adjust the hearing aid and teach basic aspects of its care and use). In most cases, the dispenser did not activate the noise management programmes nor adapted a hearing aid with adaptive noise cancellation algorithms.We believe that the aided listening subscale had relatively lower scores because few older adults participated in social activities. Thus, participating in more social activities allows people to experience a richer variety of listening environments providing a sort of listening training with the hearing aid. So the more social activities the older adults participate in while wearing the hearing aid, the better their performance in daily life listening activities, which should be reflected by better scores for the aided listening S-MARS-HA subscale .Referenceshttp://www.supersalud.gob.cl/difusion/572/w3-article-3710.html- Government of Chile [Gobierno de Chile]. Superintendencia de Salud. Problema de salud AUGE N° 56. Hipoacusia Bilateral en personas de 65 años y más que requieren uso de audífono. [Last accessed on 2019 October 16]. Available from: Page 28The sentence ending with “a better hearing aid self-efficacy” needs some editing. Perhaps “… older adults with higher educational levels exhibited higher hearing aid self-efficacy”. R. The sentence “older adults with higher educational levels could reach a better hearing aid self-efficacy” was replaced by “older adults with higher educational levels exhibited higher hearing aid self-efficacy” .Page 28, last paragraphWhen I first read “readability of the written materials” I initially thought you were referring to the S-MARS-HA but as I read on, I realized you mean the hearing aid instruction manuals. Please clarify, perhaps “readability of the written materials ”.R. The sentence was modified according to the reviewer’s comment .My co-authors and I would like to thank you and your review team for their careful reading of our manuscript and the valuable comments offered. We feel they have led to an improved version and we look forward to receiving your further advice regarding the status of our manuscript.AttachmentResponse_to_Reviewers.docxSubmitted filename: Click here for additional data file. 20 Nov 2019Does Educational Level Predict Hearing Aid Self-Efficacy in Experienced Older Adult Hearing Aid Users from Latin America? Validation process of the Spanish version of the MARS-HA questionnairePONE-D-19-19089R1Dear Dr. Fuentes-López,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: (No Response)**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This is a cross sectional observational study in a convenience sample from an urban hearing impairment outpatient program of Governmental hospital in Santiago (Chile). This paper has three components, one is the validity of a questionnaire used to measure hearing aid self-efficacy: the Spanish version of Measure of Audiologic Rehabilitation Self-Efficacy for Hearing Aids (S- MARS-HA); another second component about the role of educational level and other factors as attitudes in using hearing aids and the pure-tone average, (PTA) in the fitted ear as important factor of hearing aid self efficacy and, finally the third component of this manuscript explores the association between hearing aid self-efficacy with adherence to hearing aid use. The topic of the manuscript is appropriate for the Journal. It could be of interest to investigators and clinicians.All comments and doubts have been clarified. All recomendantions were included in the main text. So, the paper could be accepted for publishing.Reviewer #2: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. 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Subretinal prostheses are a novel technology for restoring useful vision in patients with retinitis pigmentosa or age-related macular degeneration. We characterize the surgical implantation technique and functional time window of an acute rabbit eye model for testing of human subretinal prostheses.Retinal prostheses were implanted subretinally in 26 rabbits using a two-step technique. Fundus imaging, fluorescein fundus angiography, and optical coherence topography (OCT) were conducted postoperatively from days 1 to 21 to monitor prosthesis positioning and retinal anatomic changes.Successful implantation and excellent retina apposition were achieved in 84.6% of the rabbits. OCTs showed the overlying retina at full thickness for the first 2 days after implantation. Histology confirmed intact inner layers of the overlying retina until day 3. Progressive atrophy of the overlying retina was revealed by repeated OCTs; approximately 40% of the retina thickness remained on postoperative days 5 and 6.The two-step implantation technique works well for the rabbit eye model with human prostheses. Rabbit retina may be used for acute electrophysiologic testing of a retinal prosthesis, but is unsuitable for chronic studies due to the merangiotic retina and its limited time window of validity.The improved efficacy in prosthesis surgery using this technique will circumvent the challenges in animal models that provide human-like features critical for the transition into human clinical trials. Inherited retinal diseases, such as retinitis pigmentosa (RP) and outer retinal dystrophies, including age-related macular degeneration (AMD), afflict millions of individuals causing profound vision impairment.6At present, several variations of retinal prostheses are under investigation and some are in clinical trials. Each variation differs in several ways, including material composition, number and spacing of electrodes, return electrode configuration, mechanism of light transduction to electrical stimulation, and implantation site. All devices can be categorized broadly based on the surgical implantation position with respect to the retina as either epiretinal,9The neurostimulation array of the prosthetic device used in our study was comprised of 1512 photovoltaic silicon electrodes on a 12.5-μm thick polyimide substrate.A total of 26 healthy adult New Zealand pigmented rabbits were used in this study. Mean body weight was 3.71 ± 0.55 kg. All animal studies were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Res. and were approved by the institutional animal care and use committee of University of California, San Diego. Rabbits were anesthetized with subcutaneous injections of 35 mg/kg ketamine hydrochloride and 5 mg/kg xylazine. To maintain normal body temperature, rabbits were put on a warm water blanket during the procedure. Respiratory rate, heart rate, and blood oxygen saturation were monitored continuously. To accommodate the surgical time, additional ketamine was given at half of the original dose every 30 to 40 minutes; 2.5 mg/kg xylazine was given every other redosing.For the surgical procedure, the pupil was fully dilated by 2.5% phenylephrine hydrochloride and 1% tropicamide ophthalmic solution. After induction of general and topical eye (0.5% proparacaine) anesthesia, two traction sutures (1-0 silk) were installed around the superior and inferior muscles to position the eye globe. A third traction suture also was placed in the third eyelid for better exposure of the operative field. A conjunctival peritomy was performed from 2 to 5 o'clock followed by radial cuts of the conjunctiva at 2 and 5 o'clock to create a conjunctival flap. The site of the scleral incision, 5 to 6 mm from the limbus, then was marked with a surgical marking pen. The incision was centered at 3 o'clock and was 3 mm wide. A half-thickness scleral incision was created and followed by cauterizing the choroid before full-thickness cut-through. Two polyimide glides, 3 × 35 mm and 25 μm thick, were coated in diluted Goniovisc and used to flank the prosthetic device in a sandwich fashion. The sandwich assembly then was inserted through the scleral incision into the subretinal space. The sandwich was advanced subretinally under the direct view of a surgical microscope until the neurostimulation array reached the visual streak. Subsequently, the top and bottom polyimide glides were removed before suturing the scleral incision with 7-0 sutures . IntraocAfter implantation, the eye and implant were monitored by slit-lamp, indirect ophthalmoscope, ocular tonometry, fundus imaging, optical coherence tomography (OCT), and fluorescein fundus angiography (FFA). OCT analysis was conducted on days 1, 2, 3, 5, 6, 7, 8, 14, and 21 to assess prosthesis positioning and evaluate the anatomic changes of the overlying retina over time by using the Spectralis high-resolution, spectral domain OCT imaging system . Images were obtained by line scans (6 mm) over each chip horizontally and vertically. High-quality horizontal scanning OCT images for all six chips were imported into the National Institutes of Health  software ImageJ 1.51 and retina thickness was measured after calibration. Three locations for each chip, on both ends and the middle of the chip, were chosen to measure the overlying neuroretinal thickness. Off-device retinal thickness served as normal control, which was obtained from three locations 700 to 2000 μm away from the temporal chip edge in the same horizontal scanning line. The average retinal thickness over the chips was compared to the off-device retina thickness over time. FFA was performed without anesthesia on the first postoperative day with both eyes fully dilated. The Spectralis imaging system was used for fluorescence angiography after intravenous injection of 0.1 mL 12.5% sodium fluorescein solution.The rabbits were sacrificed after a postoperative period ranging from 1 to 21 days with a lethal dose of sodium pentobarbital (120 mg/kg) under deep anesthesia . Nineteen devices were explanted for further engineering tests and analysis. Two eyes at postoperative day 1, one at postoperative day 3, and one at postoperative days 7 and 21 each were processed for histology and light microscopy. The rigidity of the device prevents paraffin sectioning and must be removed, which disturbs and lifts the overlying retina even after the eye globes have been fixed in Davidson's solution. To reduce collateral damage to the retina, transillumination was used to visualize the device head  and its t-test. P < 0.05 was considered statistically significant. Statistical analysis was performed with JMP statistical software .Retinal thickness at each location was expressed as mean ± standard deviation. The thickness of the retina over the implant and the thickness of the nearby off-device retina were compared at each time point using a paired Postoperative examinations revealed mild corneal swelling and anterior chamber reaction on postoperative day 1. Limited peripheral lens opacity was found in three rabbits without impeding fundus observation. Indirect ophthalmoscopy revealed the prosthesis in the desired position, which remained stable for all time points. The highly reflective chips were seen through the transparent rabbit retina. Some pigment changes were seen along the insertion pathway where the glides and device were introduced. Due to the unique retinal vasculature of the rabbit, the retina became very thin around day 7 and details of the subretinal array became more visually striking. An atrophic hole in the retina overlying some devices was noted upon fundus examination. FFA on day 1 showed well-demarcated hypofluorescence corresponding to the array and substrate due to the blockage of background fluorescence . Near thFFA also demonstrated less fluorescent retinal vessels on the medullary ray due to hazy optical media and silicon oil filling. Otherwise, no other noticeable findings, such as leakage and partial or total obstruction of vessels, were detected.P < 0.0001 and P = 0.0004, paired t-test). On day 3, retinal thickness decreased to 117.58P < 0.0181, paired t-test), and progressively dropped to 17.82P < 0.0001), however, the difference in retinal thickness among the individual chips on the implant was not significantly different (P > 0.05).OCT demonstrated that the position of the subretinal implants remained stable during the whole observation period. In OCT imaging, highly reflective bands with well-defined demarcation corresponded to the chips. The nontransmissible nature of the chips caused extinction of the OCT beam, which prevented Bruch's membrane and the choroid underneath the implant from being seen. The overlying retina was well attached to the chips and no subretinal fluid was present. The retina overlying the chips on postoperative days 2 and 3 demonstrated less clear layered retinal structures on OCT owing to higher tissue reflectivity. Prominent thinning and even segmental diminishing of the overlying retinal band was observed after day 5, revealing progressive retinal atrophy overlying the prosthesis on OCT . The aven day 21 . A multiOne day after implantation, histology revealed detachment of the retina overlying the implant due to the explanting procedure before histology processing. Adjacent off-device retina was normal . The oveRetinal degenerative diseases mainly involve photoreceptor degeneration as seen in RP and AMD. The inner retina, approximately 40% to 88% of inner nuclear layer cells, and approximately 20% to 30% of ganglion cells survive16Several subretinal prosthesis studies confine their electrophysiology exams to same day testing immediately postoperatively; however, it may be advantageous to record electrophysiology beyond this time point.273234In summary, our study characterized the rabbit eye model for the implantation of subretinal prosthetic devices designed for the human eye. The rabbit eye model has a time window of 2 days postoperatively for evaluation due to the nature of the rabbit's merangiotic retina. With the surgical technique described, human-sized retinal implants can be implanted into rabbit eyes with an 85% success rate. With this surgical technique and eye model, our previous work demonstrated that the recorded visual electrophysiology signals were quantitatively responsive to the engineered stimulations and provided biological feedback for further engineering optimization.Supplement 1Click here for additional data file.