Purpose. To determine whether the reduction of ganglion cell complex (GCC) thickness is uniform in the parafoveal region after vitrectomy with internal limiting membrane (ILM) peeling for idiopathic macular hole (MH). Methods. Thirty-nine consecutive eyes were studied. Vitrectomy was performed with ILM peeling with brilliant blue G (BBG) staining, and room air was used for an intraocular tamponade. The GCC thickness and retinal sensitivity were measured at the superior, inferior, nasal, and temporal quadrants around the fovea using spectral domain-optical coherence tomography (SD-OCT) and microperimetry (MP-1). The measurements were made at baseline, and at 3 and 6 months postoperatively. Results. In 38 of the 39 eyes, the MH was closed after the initial surgery. At three and six months, the percentage of eyes with significantly thinner GCC areas was higher at the temporal quadrant (40.5% at 3 months and 46.0% at 6 months) than that at the other quadrants . The retinal sensitivity was also significantly lower in the temporal area than in the other areas . Conclusion. These findings indicate that the inner retina in the temporal area may be more vulnerable to surgical manipulations than the other areas. The peeling of the internal limiting membrane (ILM) during vitrectomy has significantly improved the closure rate of macular holes (MHs) . BecauseOn the other hand, ILM peeling has been associated with alterations of the inner retinal surface, for example, a dissociated optic nerve fiber layer (DONFL) appearance , 4. OrigIn addition, a reduction in the thicknesses of the inner retina and ganglion cell complex (GCC) has been reported after ILM removal during vitrectomy for a MH . The thiA thinning of the GCC and reduction of retinal sensitivity have been reported by our laboratory. However, the results were based on measurements of the entire ILM peeled area, and the experiments were not designed to examine regional changes of the retina. In addition, the change in the thickness of macular area has been reported to continue for at least two years after ILM peeling, and the temporal retina keeps thinning but the other areas do not . TherefoIn some cases, our results showed that there were disagreements between the retinal sensitivity changes and GCC thickness. To try to resolve the lack of correspondence between the area of thinning of the GCC and the reduction of retinal sensitivity, we examined the regional changes of GCC thickness and retinal sensitivity after ILM peeling during vitrectomy for a MH. The pattern of thinning of inner retina was determined by OCT, and the associated reduction of the retinal sensitivity was determined by microperimetry.This was a retrospective, nonrandomized study of 39 eyes of 39 consecutive patients with an idiopathic MH. All cases were examined and treated at the Chiba University Hospital. This study was approved by Institutional Review Board of Chiba University Graduate School of Medicine, and the procedures used conformed to the tenets of the Declaration of Helsinki. Patients were informed on the purpose of the treatments and possible complications, and a written informed consent was obtained.The surgery was performed by three of the authors  using similar procedures. Each of the three surgeons had more than 10 years of experience in performing vitrectomy for MH closure. A 3-port pars plana vitrectomy (PPV) with a 23-gauge system was performed with a combination of phacoemulsification and aspiration and implantation of a foldable intraocular lens if the eyes were phakic. A posterior vitreous detachment (PVD) was created if one was not present. Cases with an obvious epiretinal membrane (ERM), dense cataract , glaucoma, and other retinal diseases were excluded.The ILM was made more visible by 0.25% brilliant blue G , and the ILM was gently grasped with an ILM forceps temporal to the fovea. In 6 eyes, the ILM peeling was started at the inferior retina to the fovea and these eyes were used as controls. The ILM was peeled off of the retina over approximately 3 disc diameters centered on the fovea. In all cases, an air tamponade was used and patients were instructed to maintain a face-down position for 3 days after the surgery.All patients had a complete ophthalmic examination including measurements of the best-corrected visual acuity (BCVA), microperimetry , and spectral domain-optical coherence tomography  before the surgery. Similar examinations were done at 3 and 6 months postoperatively. The data were collected and analyzed by an experienced examiner.The retinal sensitivity was determined by MP1 with the 1.4.2.SP1 version of the software embedded in the device. The Goldmann III with a 4-2 staircase strategy was used and the sensitivity of 24 locations within the central 10 degrees was determined [The GCC thickness was determined by the GCC measuring mode of the original software of the SD-OCT. The GCC thicknesses are presented as color-coded significance maps obtained by comparing the findings of the patient to the normative database embedded in the computer. We evaluated the percentage of the total area of the GCC that was significantly thinner than the embedded database in each quadrant within the central 4 mm of the significance map . The colThe outer retinal thickness was also analyzed using the software embedded in the SD-OCT. The average outer retinal thickness within the overall area is given automatically, and the baseline measurements were compared to the values at 3 and 6 months postoperatively.U test. P values <0.05 were considered to be statistically significant.Statistical analyses for the significance of differences of the GCC thickness and retinal sensitivity among the four quadrants were done with the Kruskal-Wallis test. The Mann-Whitney test was used to determine whether there were significant differences between temporal quadrant and other quadrants. The significance of the changes of the visual acuity was tested by Mann-Whitney μm and was the average of the vertical and horizontal diameters at the base of the hole. The baseline mean BCVA was 0.61 ± 0.33 logarithm of the minimum angle of resolution (logMAR) units  with a range from 0.15 to 1.22 logMAR units . The mean axial length was 23.5 ± 1.2 mm.Twenty men and 19 women were studied, and their mean age was 66.7 ± 4.3 years with a range from 58 to 75 years. The estimated duration from onset of symptoms to surgery ranged from 1 to 8 months with a mean of 2.6 ± 1.9 months. The MH stage was stage 2 in 9 eyes, stage 3 in 20 eyes, and stage 4 in 10 eyes. The mean preoperative MH size was 638 ± 300 The MH was closed in 38 cases after the initial surgery, and the remaining eye was closed after a second vitrectomy. Data from this latter eye were excluded from further analyses. In 33 phakic eyes, phacovitrectomy was performed with an implantation of intraocular lens. No intraoperative or postoperative complications, including an elevation of the IOP, were observed.P < 0.001 at 3 and at 6 months).The findings in a representative case are presented in P = 0.003 and P = 0.006). The retinal sensitivity at the temporal quadrant was lower than that at the other quadrants at 3 and 6 months . In the control eyes, the retinal sensitivity was 16.7 dB at the superior, 17.3 dB at the inferior, 17.5 dB at the nasal, and 16.1 dB at the temporal retina at 6 months postoperatively.At the baseline, the mean sensitivity at the four locations was 15.1 dB at the superior retina, 15.9 dB at the inferior retina, 15.2 dB at the nasal retina, and 15.8 dB at the temporal retina . None ofThe average retinal sensitivity decreased from 15.6 dB to 15.4 dB at the temporal retina and changed from 15.3 dB to 16.5 dB at the other three quadrants in preoperatively phakic eyes. On the other hand, the average retinal sensitivity decreased from 16.7 dB to 14.7 dB at the temporal retina and changed from 15.8 dB to 16.4 dB at the other quadrants in the pseudophakic eyes.P < 0.001, =0.001, =0.005, <0.001, resp., compared to the baseline). At 6 months, the percentage of thin areas of the GCC was 24.3 ± 23.9% in the superior, 23.4 ± 20.2% in the inferior, 11.9 ± 15.2% in the nasal, and 46.0 ± 23.0% in the temporal quadrant . The difference of the percentage of thin GCC area was significant between quadrants at 3 and 6 monthspostoperatively (P < 0.001 and P < 0.001). The percentage of thin GCC area was significantly greater in the temporal quadrant at 3 and 6 months . In the control eyes, the percentage of thin areas of the GCC was 21.9 ± 24.9% in the superior, 24.6 ± 21.6% in the inferior, 9.9 ± 18.9% in the nasal, and 52.2 ± 23.4% in the temporal quadrant at 6 months. There was a trend towards more thinning of the GCC area at the temporal retina at 6 months postoperatively although the degree of thinning was not significant (P = 0.091). This was probably because of the small number of controls.In our laboratory, the intraclass correlation coefficient of the measurements of thin GCC area is 0.917. The percentage of thin areas in the GCC was 4.7 ± 7.8% in the superior quadrant, 3.9 ± 5.8% in the inferior quadrant, 2.0 ± 7.5% in the nasal quadrant, and 2.4 ± 10.2% in the temporal quadrant at the baseline . The perμm at the baseline, 180 ± 13 μm at 3 months, and 179 ± 12 μm at 6 months postoperatively. There was no significant difference in the outer retinal thickness at 3 and 6 months compared to that at the baseline (P = 0.977 and P = 0.908).The mean outer retinal thickness was 178 ± 14 P = 0.140, R = 0.241), MH size , age , preoperative visual acuity , and axial length  at 6 months. There were no significant correlations between the temporal GCC thinned area and MH duration , MH size , age , preoperative visual acuity , and axial length  at 6 months.There were no significant correlations between the temporal retinal sensitivity and MH duration  , 11, 12.The normality of retinal function after the removal of the ILM remains controversial. The sensitivity of the retina in the area of the DONFL appearance has been reported to be not significantly different from the area without a DONFL , 13. HowThe reduction of the GCC thickness and retinal sensitivity after ILM peeling was most severe at the temporal quadrant. The reason for this restricted change to the temporal retina might be as follows. First, the removal of the ILM is started from the temporal retina to the fovea because we had assumed the temporal retina was safer to start the peeling of the ILM because the terminals of retinal nerve fibers exist at temporal retina. Although the surface of retina was gently grasped by forceps to try not to damage the retina, this might have affected the temporal GCC thickness. Second, the nerve fiber layer has been reported to be thinnest in temporal quadrant around fovea . In addiBBG was used to make the ILM more visible. The ILM is transparent, and different agents including indocyanine green, trypan blue, BBG, autologous serum, and triamcinolone acetonide have been used to make it more visible. Some of these agents have been reported to be toxic to retinal neurons and can be responsible for the reduction of GCC. However, we suggest the influence of dye is minimal because BBG has been reported to be cytoprotective to retinal neuronal cells , and we The major limitations of this study are the small number of eyes and the short postoperative observation period. The number of cases was relatively small because this study was conducted at a single institution. However, the surgical procedures were similar, and the collection of data was well organized. Three skilled surgeons with comparable experience of more than ten years performed surgeries. Although the ILM peeling area and its symmetry were not completely similar between cases, the peeling area was wide enough to cover the entire studied area. The observation period was six months and a longer postoperative period is needed because the thinning of the entire retinal thickness has been reported to continue for up to 24 months . Even soIn conclusion, we have investigated the regional changes of the GCC thickness and retinal sensitivity after ILM removal during surgery for an idiopathic MH. The findings that the GCC at the temporal quadrant is thinner and the retinal sensitivity at the same area was lower suggest that the inner retina at the temporal area may be more vulnerable to the surgical procedures.


Critical Care, Heinrich and colleagues reported that etomidate for anesthesia induction had similar outcome in comparison with other drugs, in a specific population of 3,054 patients ahead of major cardiac surgery. For the authors, the similar outcomes for mortality and length of hospital stay add to the hemodynamic stability and the improved intubation conditions to support keeping etomidate in the emergency drugs armamentarium for induction of anesthesia in patients at risk of hemodynamic failure. This commentary reviews the results and implications of their study.Use of etomidate for anesthesia induction is still debated. In the previous issue of  Etomidate is often considered as a two-edged sword and its use for induction of anesthesia is thus controversial. On the one hand, etomidate is used for its hemodynamic stability after anesthesia induction and also allows good intubation conditions, especially in more severely ill patients, and on the other it has negative effects on steroid synthesis. The relative importance of both the positive and negative effects for outcome of patients in both anesthesia and/or critical care is, however, a highly controversial issue.Critical Care, Heinrich and colleagues [In the previous issue of lleagues  reportedThe results of Heinrich and colleagues’ retrospective study are concordant with the recent study of Wagner and colleagues , a retroEtomidate use for anesthesia induction has been an important subject of controversy over the past years. Indeed, it has been well known that adrenal mitochondrial 11-β-hydroxylase activity is transiently inhibited by a single dose of etomidate, with consecutive adrenal suppression . WhetherCardiac surgery and the initiation of cardiopulmonary bypass are stimulators of the inflammatory response. Variations in cortisol occur after major cardiac surgery and because cortisol acts synergistically with endogenous epinephrine and norepinephrine, the impairment of these stress hormones after etomidate induction is likely to contribute to an increase in vasopressor requirement. In the current study , the perPost-induction hypotension has been associated with increased morbidity and mortality, both in anesthesia  and inteThe similar outcomes observed in this study  for mortSome limits of the study  have to Nevertheless, Heinrich and colleagues  underlinThere are no convincing or consistent data from evidence-based medicine that etomidate is associated with poor outcome, especially increased mortality, particularly if adjustment is made for pre-existing severity of illness. Although some authors have suggested that, in the ICU, physicians should abandon the use of etomidate, the present study of Heinrich and colleagues , perform


Background. The aim of this study was to analyze the effect of vestibular schwannoma microsurgery via the retrosigmoid-transmeatal approach with special reference to the postoperative tinnitus outcome. Material and Methods. A prospective study was performed in 89 consecutive patients with unilateral vestibular schwannoma indicated for microsurgery. Patient and tumor related parameters, pre- and postoperative hearing level, intraoperative findings, and hearing and tinnitus handicap inventory scores were analyzed. Results. Cochlear nerve integrity was achieved in 44% corresponding to preservation of preoperatively serviceable hearing in 47% and useful hearing in 21%. Main prognostic factors of hearing preservation were grade/size of tumor, preoperative hearing level, intraoperative neuromonitoring, tumor consistency, and adhesion to neurovascular structures. Microsurgery led to elimination of tinnitus in 66% but also new-onset of the symptom in 14% of cases. Preservation of useful hearing and neurectomy of the eighth cranial nerve were main prognostic factors of tinnitus elimination. Preservation of cochlear nerve but loss of preoperative hearing emerged as the main factor for tinnitus persistence and new onset tinnitus. Decrease of THI scores was observed postoperatively. Conclusions. Our results underscore the importance of proper pre- and intraoperative decision making about attempt at hearing preservation versus potential for tinnitus elimination/risk of new onset of tinnitus. Subjective tinnitus is a false perception of sound in the absence of an acoustic stimulation. It is the second most frequent symptom among patients with vestibular schwannoma, occurring in 63–75% of patients . In abouThe mechanisms of tinnitus origin in vestibular schwannoma are complex. Several potential mechanisms of tinnitus generation have been suggested in the literature  , 5.There are a limited number of studies carried out on postoperative occurrence of tinnitus in both hearing preserving and hearing nonpreserving vestibular schwannoma microsurgery –15. MoreWith all the advances in diagnostics and therapeutic techniques leading to reduction of morbidity and mortality, the debate on vestibular schwannoma management has centered largely on the quality of life issues. Consequently, postoperative tinnitus has increased in importance as a factor affecting the patient's quality of life .This study analyzed the effect of vestibular schwannoma resection via the retrosigmoid-transmeatal approach on postoperative tinnitus. The analyses were made with special reference to the effect on tinnitus with attempted hearing preservation. In light of these results, we discuss the rationale for attempting to preserve hearing when surgically treating vestibular schwannomas.A prospective study was performed that included all 89 consecutive patients with untreated unilateral sporadic vestibular schwannoma indicated for surgical treatment via a retrosigmoid-transmeatal approach in the period from January 2008 to December 2010. All patients were operated on by the same team of two neurotologists and a neurosurgeon. Design of the study was approved by the local ethical committee.Details of the surgical technique were reported elsewhere ; thus, oThe data collected in each patient included the patient's age, gender, size of tumor, pre- and postoperative hearing level, intraoperative findings , and perioperative complications. Validated questionnaires (Hearing Handicap Inventory for Adults (HHI) and Tinnitus Handicap Inventory (THI)) were employed for assessment of pre- and postoperative symptoms.Size of the tumor was based on preoperative magnetic resonance imaging (MRI). The diameter was measured on the axial scans in the plane parallel with the long axis of the internal auditory canal (IAC) including both intra- and extrameatal portion of the tumor. Furthermore, Koos grading system was used to classify the tumor grade based on tumor extension . Postoperative MRI scan to exclude tumor recurrence was undertaken at 3, 12, and 48 months postoperatively.Facial nerve function was assessed according to the House-Brackmann grading system at discharge and on the last follow-up control (29–64 months postoperatively).Hearing level was assessed according to the pure tone audiogram with measurement of pure tone average (PTA) and speech discrimination score (SDS). It was graded according to Gardner-Robertson's classification (GRC) . GRC1 and GRC2 were accepted as useful hearing level.We have analyzed the results of subjective audiometric tests ; speech audiometry parameters: speech discrimination score (SDS) and speech reception threshold (SRT)). To evaluate the results of BAEP, we used the Hannover classification .The extent of tumor extension into the internal auditory canal was evaluated on preoperative T2-weighted MRI. The presence of fluid lateral to the tumor was considered as partial filling while absence was considered as complete filling.The intraoperative findings that were analyzed included bleeding, tumor consistency, adhesion to neurovascular structures, presence of cystic component, and success in identification of cochlear portion of the eighth cranial nerve. t-test, Tukey HSD test, and one-way ANOVA as appropriate. Analyses were performed using SPSS v16.0 .Results were analysed using chi square test, Fisher's exact test, Student'sAll tumors were removed radically . We encountered 1 case of tumor recurrence on the MRI at 48 months following surgery affecting labyrinth and internal auditory canal. It was managed with stereoradiosurgery. In all the remaining cases, patients are disease-free.There were neither infectious complications including meningitis nor perioperative mortality. The loss of continuity of the facial nerve occurred in 6% (5/89) of patients. In all cases, a direct reconstruction of the facial nerve in the cerebellopontine angle/internal auditory canal led to reinnervation (House-Brackmann grade III function). In the remaining 94% (84/89) of cases, facial nerve was preserved. Immediate postoperative excellent or very good function of the facial nerve (House-Brackmann grades I and II) was observed in 65% (58/89) of the patients. At the last follow-up, we observed excellent or very good function of the facial nerve in 67% (60/89) of cases while the severe dysfunction (House-Brackmann grade V) was present in 1% (1/89) of patients.P < 0.01) and stage of the tumor (P < 0.01) , success rate of serviceable hearing preservation in such cases was only 47% (17/36). Overall hearing was preserved in 21% (17/81) of cases with preoperatively serviceable hearing . In 79% (64/81) of patients, we demonstrated postoperative deafness . Success rate of hearing preservation was dependent on the size ( < 0.01) . Level oP < 0.01) . Also, the type of BAEP response was related to the hearing preservation (P = 0.03) and possibility to employ intraoperative BAEP was crucial for the outcome (P < 0.01). Surprisingly, preservation of BAEP response intraoperatively did not correlate with hearing preservation.Preservation of preoperatively useful hearing was demonstrated in 21%  and the nonuseful hearing in 29%  of the patients. Moreover in two patients we observed postoperative improvement of hearing from the nonuseful to useful level. Success rate of hearing preservation was dependent on the level of preoperative hearing ( < 0.01) . AccordiP < 0.01), soft consistency of the tumor (P = 0.05), and the absence of adhesion of tumor to neurovascular structures (P < 0.01). Bleeding or presence of cystic components did not affect the success rate of hearing preservation.Among the key intraoperative factors affecting the chance for hearing, preservation belonged to identification of the cochlear nerve (P = 0.03). Significant differences in the mean postoperative HHI level across the studied groups were not observed .HHI scores were correlated with the hearing level as assessed according to Gardner-Robertson's classification. Preoperatively, the mean HHI levels were lowest in the group of patients with useful hearing (GRC1-GRC2: 12 ± 16), followed by the group of patients with nonuseful hearing (GRC3-GRC4: 24 ± 22) and patients with nonuseful hearing/deafness (GRC5: 34 ± 17)  of the patients. It was slightly more prevalent in cases with preoperatively deafened ears. As such, tinnitus was reported by 88% (7/8) of patients with GRC5 while among patients with serviceable hearing it was reported in 82% (67/81). In case of preoperatively useful hearing tinnitus was observed in 74% (32/43) and in case of nonuseful hearing in 90% (34/38) of the patients .Tinnitus was reported by 35% (29/89) of our patients postoperatively . Among pIn relation to the preoperative hearing level, postoperative tinnitus was more prevalent in patients with preoperatively serviceable hearing. It was reported by 35% (15/43) of patients with preoperatively useful hearing, by 34% (13/38) of patients with nonuseful hearing, and by only 12% (1/8) patients with preoperatively deafened ear .P = 0.05) (Perception of postoperative tinnitus was correlated to the status of preservation of the cochlear nerve and postoperative hearing level ( = 0.05) . HighestWhen analyzing the new onset of tinnitus, 75% (3/4) of reported cases were observed in patients with postoperative deafness and the remaining case was observed in the group of preserved nonuseful hearing . We did P = 0.02) (Results were correlated to the success rate of cochlear nerve and hearing preservation ( = 0.02) . We did Elimination of tinnitus was observed in 75% (6/8) of preoperatively deafened, in 61% (23/38) of nonuseful hearing, and in 44% (19/43) of useful hearing ears. If correlated with postoperative hearing level, tinnitus disappeared in 51% (37/72) of GRC5, 63% (5/8) of GRC3-GRC4, and 67% (6/9) of GRC1-GRC2.P < 0.01)  . HighestP = 0.01 and P = 0.03, resp.).When THI scores were correlated with the hearing level as assessed according to Gardner-Robertson's classification, we did not observe any differences of THI scores across the studied groups preoperatively  and postoperatively . There was no relationship between the THI and HHI scores in all the studied groups pre- and postoperatively. Overall microsurgical treatment of vestibular schwannomas led to improved quality of life as related to impact of tinnitus as the mean THI scores decreased from preoperative to postoperative level (preoperative THI: 18 ± 20 versus postoperative THI: 14 ± 19). Nevertheless, the worst THI scores over 50 were observed only among patients who lost preoperative hearing and became deaf despite the cochlear nerve preservation as opposed to those who had eighth nerve neurectomy or hearing preserved . In agreement with the published literature, main prognostic factors for hearing preservation were size of the extrameatal portion and stage of the tumor, preoperative hearing level, and possibility to employ intraoperative BAEP monitoring. We did not confirm the observation that level of internal auditory canal filling is crucial for the result. Besides that, tumor consistency and adhesion to neurovascular structures were important predictive factors for the result.Concerning the HHI scores, observed handicap was correlated to the hearing level preoperatively. Postoperatively, we observed increase of handicap in all patients irrespective of the result. It is interesting that the subjectively perceived hearing handicap postoperatively was not different for the studied groups (useful hearing versus nonuseful hearing versus deafness).We have unpublished results on patients from the same group who were rehabilitated postoperatively for the single sided deafness. In this group of patients who have decided on BAHA according to the soft-band testing and Bern benefit test, the HHI scores were lowest following the implantation. It is striking that scores were significantly better in the postrehabilitation period when compared to both postoperative period with single sided deafness and preoperative period when serviceable or even useful hearing was present. This could be at least partly explained by the alteration of sound localization and distortion leading to impaired speech intelligibility as a consequence of dysfunction of inner ear and cochlear nerve by tumor and its treatment , 36.Tinnitus is a bothersome symptom for patients with vestibular schwannoma that appears to influence quality of life , 13. ItsOverall, in our study of patients undergoing vestibular schwannoma microsurgery via retrosigmoid-transmeatal approach, we observed disappearance of preoperative tinnitus in 66% but also new onset of the symptom in 14% of patients. We did not observe any relation with the tumor size, age, and gender of the patients. There was a trend for association of worse preoperative hearing with preoperative tinnitus. Such results correspond to some of the previously published works when hearing preserving surgery was attempted , 13.Both preservation of useful hearing and neurectomy of the eighth cranial nerve emerged as the main prognostic factors of tinnitus elimination with microsurgery. On the contrary, preservation of cochlear nerve but loss of preoperative hearing was identified as the main factor for both tinnitus persistence and new-onset tinnitus. This is in contrast to the study of Kameda et al.  who in tAs we did not encounter any case of the newly perceived tinnitus in patients with preoperative deafness when there was no attempt at the eighth cranial nerve preservation, we support such cases hearing/eighth cranial nerve nonpreserving surgery. Similar results were achieved in cases of useful hearing preservation but such result is hard to predict intraoperatively.As all cases of new tinnitus occurred when attempting to preserve cochlear nerve and hearing but finally hearing deteriorated to deafness or nonuseful level, we expect important role of inner ear and cochlear nerve in the pathophysiological mechanisms of its origin. Such consequences were correlated with the worst THI scores as opposed to handicap improvement when tinnitus was alleviated with surgery. In contrast to our results, del Río et al.  in theirAdequate preoperative discussion of potential impact of tinnitus on quality of life with vestibular schwannoma microsurgery indicated for patients seems to be crucial as well. In general severity of tinnitus correlates to psychological and general health factors and the risk of depression and insomnia may be higher in patients with tinnitus .This study analyzed the effect of vestibular schwannoma microsurgery via the retrosigmoid-transmeatal approach with special reference to the effect on tinnitus with attempted hearing preservation. Our results show that despite the fact that the cochlear nerve integrity can be preserved in a significant number of patients even in large tumors hearing preservation is achieved only in selected cases. Main prognostic factors predicting hearing preservation are extent of tumor, preoperative hearing level, possibility of intraoperative neuromonitoring, tumor consistency, and adhesion to neurovascular structures. Overall tumor removal led to elimination of tinnitus in a significant proportion of patients. Preservation of useful hearing and neurectomy of the eighth cranial nerve emerged as the main prognostic factors of tinnitus elimination. On the contrary, preservation of cochlear nerve but loss of preoperative hearing was identified as the main factor for both tinnitus persistence and new-onset tinnitus. Our results underscore the importance of proper pre- and intraoperative decision making about attempt at hearing preservation versus potential for tinnitus elimination/risk of new onset of tinnitus.


TCs) are a novel type of interstitial cells present in a wide variety of organs and tissues (www.telocytes.com). Telocytes are identified morphologically by a small cell body and specific long prolongations (telopodes) alternating thin segments (podomers) with dilations (podoms). The presence of TCs in rat meninges has been identified in previous research. We here present further evidence that TCs existed in canine dura mater, closed to capillary and surrounded by a great deal of collagen fibres under transmission electron microscope.Telocytes ( Telocytes present in rat meninges and choroid plexus were firstly reported by Popescu's group Telocyte (TC), a novel characteristic stromal cell, was firstly described in 2010 by Popescu's group as a case of serendipity Three male beagles  were anaesthetized with diazepam  and ketamine hydrochloride  by intramuscular injection. The anaesthetized animals were fixed in the supine position with their necks extended and thoracic cavities were opened, through a puncture in the left ventricle; 2500 ml of heparin physiological saline and 1000 ml 4% formaldehyde solution each animal were perfused at physiological pressure, respectively. After the perfusion, dura mater was removed. Transmission electron microscope was performed on small tissue fragments, processed according to routine Epon‐embedding procedure. And the processed specimen were observed under TEM , as previously described by Gherghiceanu and Popescu Telocytes with typical long and thin prolongations (Tps) were fundamentally identified in canine dura mater under TEM Figs , 2, 3, 4The most distinct property of TCs is the existence of very long, thin and moniliform prolongations, termed Tps. So far, although no specific markers have been yet defined for TCs, the immunostaining with CD34/PDGFR‐alpha The authors confirm that there are no any conflicts of interest.


Initially described as an early onset seizure variant of Rett syndrome, the CDKL5 disorder is now considered as an independent entity. However, little is currently known about the full spectrum of comorbidities that affect these patients and available literature is limited to small case series. This study aimed to use a large international sample to examine the prevalence in this disorder of comorbidities of epilepsy, gastrointestinal problems including feeding difficulties, sleep and respiratory problems and scoliosis and their relationships with age and genotype. Prevalence and onset were also compared with those occurring in Rett syndrome.Data for the CDKL5 disorder and Rett syndrome were sourced from the International CDKL5 Disorder Database (ICDD), InterRett and the Australian Rett syndrome Database (ARSD). Logistic regression (multivariate and univariate) was used to analyse the relationships between age group, mutation type and the prevalence of various comorbidities. Binary longitudinal data from the ARSD and the equivalent cross-sectional data from ICDD were examined using generalized linear models with generalized estimating equations. The Kaplan-Meier method was used to estimate the failure function for the two disorders and the log-rank test was used to compare the two functions.The likelihood of experiencing epilepsy, GI problems, respiratory problems, and scoliosis in the CDKL5 disorder increased with age and males were more vulnerable to respiratory and sleep problems than females. We did not identify any statistically significant relationships between mutation group and prevalence of comorbidities. Epilepsy, GI problems and sleep abnormalities were more common in the CDKL5 disorder than in Rett syndrome whilst scoliosis and respiratory problems were less prevalent.This study captured a much clearer picture of the CDKL5 disorder than previously possible using the largest sample available to date. There were differences in the presentation of clinical features occurring in the CDKL5 disorder and in Rett syndrome, reinforcing the concept that CDKL5 is an independent disorder with its own distinctive characteristics. DKL5) gene [The CDKL5 disorder is a rare X-linked neurodevelopmental disorder caused by a disruption in the cyclin dependent kinase-like 5 (CL5) gene . MutatioL5) gene , 2. LateL5) gene . TherefoL5) gene . Less coL5) gene .Rett syndrome is a well-studied rare neurodevelopmental disorder caused by a mutation in the methyl CpG binding protein 2 (MECP2) gene . Like thUnlike Rett syndrome, very little is known about the full range of comorbidities occurring in the CDKL5 disorder with available literature mainly limited to small case series or studies –3, 16–32http://cdkl5.childhealthresearch.org.au) was established in 2012 with the purpose of collecting information internationally on individuals with the CDKL5 disorder. A CDKL5 disorder specific questionnaire was formulated to capture comprehensively the clinical features and natural history of the disorder [CDKL5 mutation was considered to be pathogenic or probably pathogenic and when at least one relevant section of the questionnaire had been completed. As the CDKL5 disorder was initially considered to be an atypical form of Rett syndrome, data on individuals with the CDKL5 disorder were initially included in InterRett, an international phenotype database that collects data worldwide on the clinical features and genetic characteristics of individuals with Rett syndrome [Data for the CDKL5 disorder were sourced from the International CDKL5 Disorder Database (ICDD) with supplementation of a small number of cases from InterRett, the International Rett Syndrome Database. The ICDD , a longitudinal database that collects information from families and clinicians . QuestioEthics approval for this study was provided by the University of Western Australia Human Research Ethics Committee (RA/4/1/5024).The covariates included age group, gender, mutation type and for comparative purposes the type of disorder (CDKL5 disorder and Rett syndrome). Age at registration to ICDD was grouped into three categories  consistent with the age distribution of the study sample, which comprised mainly of young children and few adults . Due to the large number of different individual CDKL5 gene mutations and the few recurrent mutations, the mutation types were categorized as previously  accordinAge of onset and current seizure frequency  were used to characterise epilepsy. Parents were asked to report whether their child had ever and currently had constipation, reflux and air swallowing; the method of feeding ; ability to self-feed  restricted to children aged 2 years or more, the occurrence/frequency of coughing during meals for those who fed orally; and the age at gastrostomy insertion. They were also requested to report on the presence of any sleep problems and specific problems of night waking and night laughing, and to complete the Sleep Disturbances Scale for Children (SDSC). The SDSC is a 26 item self-report scale, which allows parents to rate their children’s sleep behaviour using a 5-point Likert scale during the previous 6 months . InformaInformation was available on age of onset of epilepsy, and gastrostomy insertion; and the presence of sleep problems, gastrointestinal problems and irregular breathing patterns at various time-points in the life of the individual. The occurrence of lower respiratory tract infections over the life course prior to the initial follow-up questionnaire and during previous 12-month periods for subsequent follow-up questionnaires was also available. Recently published information was used for estimates of age of onset of scoliosis .CDKL5 disorder and Rett syndrome. Logistic regression (multivariate and univariate) was used to analyse the associations between age group, mutation type and the prevalence of various comorbidities. Multinomial logistic regression was used to investigate the relationship between the frequencies of coughing during meals and the independent variables. Odds ratios and beta coefficient values were examined to interpret the effect sizes. Sleep scores (SDSC) of individuals with CDKL5 disorder were compared with a normal population sample [t-test with unequal variance. The incidence rate of hospitalisation (in 100 person-years) was determined using the number of hospitalisations over the total person-time of the hospitalised individuals.Descriptive statistics were used to characterise the comorbidities  in individuals with n sample  using twThe comparison with Rett syndrome was restricted to females for both disorders such that males with the CDKL5 disorder were excluded. Binary longitudinal data from the ARSD and the equivalent cross-sectional data from ICDD were examined using generalized linear model with generalized estimating equations. This accounted for clustering within individuals, to estimate the effect of the type of disorder on the likelihood of selected comorbidities after adjusting for age. Log link function, robust standard errors and exchangeable working correlation structures were used in the model for parameter estimation. For time to event analysis, the Kaplan-Meier method was used to estimate the failure function for the two disorders and the log-rank test was used to compare the two functions. Event time was defined as the age at onset of scoliosis, epilepsy or gastrostomy or the age of ascertainment if censored. STATA  was used for statistical analysis.CDKL5 mutation registered either with ICDD (n = 151) or InterRett (n = 16). Age at registration ranged from 3.9 months to 29.1 years (median age 5.9 years). The majority  were females  and 14.4 % (n = 24) were males . Sufficient mutation details were available for 164 cases to enable classification into mutation groups based on the structure and function of the CDKL5 gene. Case distribution, according to gender, age group, mutation group and comorbidities is shown in Table As of June 2015, there were 167 confirmed cases with a pathogenic n = 82), the majority (56/82) were experiencing fewer than five seizures a day, 18/82 between five and ten seizures, and 8/82 more than 10 seizures every day. Otherwise, almost 13.9 % (19/137) were having seizures approximately weekly, 9.5 % (13/137) monthly and one individual (0.7 %) only occasionally experienced seizures. Nine individuals (6.6 %) had had no seizures for more than a year.Families of all  but two females and one male reported that their child had experienced one or more episodes of seizures. The median age of epilepsy onset was 6 weeks (range 1 day to 1.5 years). Information on current seizure frequency was available for 137/145. Ninety-five individuals  were experiencing seizures daily with their average frequency of daily seizures ranging from one to 21 seizures. Of those who provided information on the number of daily seizures  of the individuals were reported to have experienced at least one gastrointestinal (GI) problem including constipation (70.9 %), reflux (64.1 %) and air swallowing (27.1 %)  were reported to eat and drink orally. Of these, 12 children had had a feeding tube inserted for other purposes such as venting gas and administration of nutritional supplements or medications. Twenty-eight individuals (20.7 %) were exclusively enterally fed (gastrostomy tube or nasogastric tube feeding). Use of nasogastric tube feeding was uncommon .Feeding difficulties were reported for over a half 50.9 %, 48/95) of the orally fed individuals with 60 % (45/75) of the children over 2 years of age completely dependent on their families for eating and drinking. A much smaller number  of children were able to eat/drink independently; some  could finger feed with assistance or were assisted with spoon-feeding . Almost three-quarters  of the parents reported at least occasional (>1/week) coughing during oral feeding. There were some evidence to suggest that the relative risk of coughing (sometimes or often vs never) during feeding was greatest in those under 5 years  of individuals had had a gastrostomy or jejunostomy tube inserted due to various and sometimes multiple reasons including feeding difficulties (80.0 %), poor weight gain (60.0 %), difficulty with administration of medications (62.9 %) and poor general health and wellbeing (51.4 %).p = 0.013) with a quarter of males having gastrostomy insertion by age 2.6  compared to 7.4 years  for females .Time to event analysis showed that males were more likely to undergo gastrostomy than females  of individuals were reported to have experienced a sleep problem during their lifetime with current night waking occurring in 58.5 % (72/123)  [p < 0.001)   Fig. 2)t-test wiAlmost one-third 32.5 %, 41/126) were reported to have breathing irregularities at the time of registration. These included hyperventilation  and breath holding   of other acute illnesses in 5-year intervals over their child’s lifetime Table . During Seizure-related hospitalisations accounted for nearly two-thirds  of these hospitalisations over 706.5 person-years with an incidence of 47.4 admissions per 100 person-years. For the children of the 69/91 families with seizure-related admissions who provided sufficient detail on these, the mean number of days in hospital due to seizure-related events was 27.4 . Respiratory problems including aspiration, pneumonia, bronchitis and bronchiolitis accounted for 62 hospitalisations (11.7 %). The incidence of respiratory-related hospitalisations was 8.2 admissions per 100 person-years. Thirty-seven  individuals had had at least one hospitalisation related to respiratory problems over their lifetime.The risk of developing scoliosis increased with age with a 68.5 %  likelihood of developing scoliosis by age 10 years , 13.8 years (2.8–27.3), 15.0 years (2.3–29.1), 15.8 years (2.6–30.8), 17.9 years (3.0–34.2) and 17.8 years (2.6–35.5) for the questionnaire years 2000, 2002, 2004, 2006, 2009 and 2011 respectively.Follow-up data at one or more collection points were available in the ARSD for 321 females with a pathogenic p <0.001) during the observation period.The median age of epilepsy onset was much earlier in females with the CDKL5 disorder (6.0 weeks) than Rett syndrome 4.9 years)  . The median age of scoliosis onset for females with the CDKL5 disorder  was much later than in Rett syndrome  [Sleep problems were highly prevalent in both disorders but while night waking was more prevalent in the CDKL5 disorder than Rett syndrome, night laughing was less prevalent as were gastrointestinal problems (except for reflux), and autonomic-type breathing problems Table . However0, 11.6)  This large-sample study using a CDKL5 disorder specific database is the first to examine the comorbidities of the CDKL5 disorder in depth and to compare their occurrence with that of Rett syndrome. Epilepsy, GI problems and sleep abnormalities were common in the CDKL5 disorder whilst scoliosis and respiratory irregularities were less prevalent than in Rett syndrome. The likelihood of experiencing epilepsy, GI problems, respiratory problems, and scoliosis increased with age and males were more vulnerable to respiratory and sleep problems than females. There was no clear relationship between mutation group and prevalence of comorbidities. However, mutations associated with late truncations after aa781 seemed to play some protective role against GI problems, autonomic breathing irregularities and sleep problems. It was also observed that there were differences in clinical features occurring in CDKL5 disorder and Rett syndrome, reinforcing the notion that CDKL5 is an independent disorder with its own distinctive characteristics.Consistent with previous studies, the early onset of epilepsy (median age of onset 6 weeks) was a key feature of CDKL5 disorder , 20. Oven = 4 and n = 10) where all subjects were observed to have experienced some sleep abnormalities including disorders of initiation and maintaining of sleep, sleep breathing disorders and daytime somnolence. In our study, the Sleep Disturbance Scale for Children was used to assess the various aspects of sleep disturbances in a large sample with CDKL5 disorder for the first time. When compared to normal healthy children [Our large sample size has allowed us to characterize sleep difficulties in the CDKL5 disorder in much greater depth than was possible in previous small case series , 27. Nigchildren , the frechildren –44. Evenchildren , 45, 46.children . Perhapschildren .n = 8) and apneustic breathing (n = 2) types was present in all the ten females [Autonomic breathing irregularities including breath holding and hyperventilation occurred in approximately one third of our CDKL5 affected sample (32 %) compared with 15/20 females in the French clinical study . In an I females . In the  females . The dif females , 24. TheLower respiratory tract infections including pneumonia were reported for over one fifth of the CDKL5 affected individuals. This is an important issue especially among the younger children that might relate to feeding and swallowing difficulties, aspiration or difficulties in expectorating chest secretions due to a poorly effective cough. Aspiration of food or fluids into the lungs was reported for nearly a quarter of the affected individuals. Over a quarter were also unable to cough up chest secretions, which might be a contributing risk factor for aspiration because coughing is a protective mechanism to prevent aspiration .Approximately one third of individuals would have developed scoliosis by the age of 10 years. In comparison to other comorbidities, scoliosis was the least frequently observed. The occurrence of scoliosis has been rarely reported previously in the literature, possibly due to the younger age distributions in these studies. A study by Artuso et al.  reportedAge can be considered as a risk factor for comorbidities such as epilepsy, GI and sleep problems, respiratory issues and scoliosis. The likelihood of each of these comorbidities increased with age except for the frequency of coughing during feeding. Younger children would be more prone to coughing as their small and narrow airways get obstructed easily . Over tiThe influence of gender on the prevalence of various comorbidities was clearly evident. As generally reflected in our data, males were more vulnerable to various respiratory and sleep problems. X-inactivation (XCI) and different degrees of skewing in females may result in variable expression of CdKL5 protein, which may improve the expression of the phenotype in females. XCI is the transcriptional inactivation of one of the two X chromosomes in females to functionally equalize the gene dosage imbalance of X linked genes between XX females and XY males . Our recn = 4) had better walking and hand use ability and less frequent refractory epilepsy compared with those with mutations in the kinase domain and frameshift mutations in the C-terminal region (n = 7) who had a severe phenotype including infantile spasms, frequent refractory epilepsy, severe motor impairment and inability to walk.Even though no significant relationship between mutation type and various comorbidities was found, there were consistencies in the distribution of comorbidity status by mutation group. For instance, there was some evidence that mutations associated with late truncations after aa781 may play a protective role against some GI problems, autonomic breathing irregularities and sleep problems compared to those with no function protein. Individuals with missense/in-frame mutations within the catalytic domain appeared to have higher odds of having any GI problems  and autonomic breathing irregularities (hyperventilation and breath-holding) when compared to those with no functional protein. We previously showed that late truncation mutations after aa781 were generally associated with a milder phenotype and had attained better gross motor, hand function and communication skills than those with other mutations . Some ge5 disorder than in Rett syndrome (4.9 years). As reflected in our time-to-event curve (Fig. There have been several studies , 32, 52 syndrome .9 years.Gastrointestinal problems except for reflux were slightly less prevalent in CDKL5 affected females than Rett syndrome. Rett syndrome is generally associated with a high prevalence of gastrointestinal dysmotility, feeding difficulties and poor growth , 54, 55.Sleep problems including night waking, screaming and laughing were more likely to occur in the CDKL5 disorder than Rett syndrome. The likelihood of experiencing a sleep problem in the CDKL5 disorder increased with age. The reason for this is not clear but it could relate to the persistent and refractory epilepsy. Our findings also suggest that autonomic dysfunction is less affected in CDKL5 disorder than in Rett syndrome. On the other hand, lower respiratory tract infections such as pneumonia, were a little more likely to occur in CDKL5 affected females than in those with Rett syndrome. This might be due to increased occurrence of reflux and feeding difficulties in CDKL5 disorder contributing to increased risk of aspiration.CDKL5 mutation to date therefore enabling greater precision in our statistical estimates. This is the first time a CDKL5 specific questionnaire has been used to investigate the comorbidities of the disorder. Engagement of the consumer reference group in the development of the questionnaire also gives strength to this study. The ICDD is growing and in time will provide an even larger data repository for the better understanding of various aspects of the CDKL5 disorder. In addition, compared to the previous case series and case studies, the age range of the individuals in the CDKL5 registry is wide providing a better estimate of the prevalence and characteristics of comorbidities, particularly those that develop with time.Our study comprised the largest number of individuals with a confirmed pathogenic 5 disorder is a condition that requires expensive genetic testing for diagnosis, selection bias according to socioeconomic status may be occurring. Families from higher socioeconomic backgrounds are more likely to have the financial capacity or insurance to cover the costs of genetic testing, especially in countries such as USA where the socioeconomic gradient is marked. This may result in many individuals with a CDKL5 mutation remaining undiagnosed and thus excluded from the registry. This is a cross sectional data collection, and longitudinal data collection similar to Rett syndrome [There were some limitations in this study that might impact the results. As the CDKL5 disorder questionnaire asks parents to provide information of their child’s condition since birth, there is a possibility for some recall error. To counter this, parents were asked to refer to their child’s medical records and diaries before the completion of questionnaire. Families did provide very extensive medical histories about their children but we acknowledge this was from a patient rather than a clinical perspective. Another limitation is that, a complete record of information was not available from every confirmed case, as parents may have completed different sections, resulting in varying denominators for various outcomes. Since the CDKLsyndrome  would beThis study provides a comprehensive picture of comorbidities in the CDKL5 disorder. Being the largest sample to date, the assumptions and results obtained gives a much more precise insight into the phenotypic expression of this disorder than was previously possible. Epilepsy, gastrointestinal and sleep problems were shown to be extremely prevalent, whilst scoliosis and respiratory problems were relatively less so. We have demonstrated that the impacts of daily feeding and sleeping difficulties in CDKL5 disorder both for the child and their family are issues of concern in urgent need of further clinical research. There is also a need to evaluate quality of life both for the child and their family and for this to be considered as an important outcome when investigating optimal strategies for managing the many complexities of this disorder. Finally it is heartening to know that the growing size of the CDKL5 registry will provide a valuable platform for further future study of genotypic-phenotypic relationships in this disorder.


Zip4, in the cochleae of mice treated with M+V. Immunohistochemistry demonstrated an intense Zip4 signal in cochlear tissues such as the lateral wall, organ of Corti, and spiral ganglion. Finally, mice treated with the Zip4 inducer (–)-epigallocatechin-3-O-gallate showed a significant reduction in the increase of the ABR threshold at 32 kHz and up-regulation of Zip4 expression in the cochlea. This study suggests that epigenetic regulatory pathways can modify auditory function and that zinc intake in the cochlea via Zip4 mediates maintenance of mammalian hearing.Various factors that are important for proper hearing have been identified, including serum levels of zinc. Here we investigated whether epigenetic regulatory pathways, which can be modified by environmental factors, could modulate hearing. RT-PCR detected expression of genes encoding DNA methyltransferase and histone deacetylase (Hdac) in the postnatal as well as adult mouse auditory epithelium. DBA/2J mice, which are a model for progressive hearing loss, were injected subcutaneously with one or a combination of the following reagents: <smallcaps>L</smallcaps>-methionine as a methyl donor, valproic acid as a pan-Hdac inhibitor, and folic acid and vitamin B12 as putative factors involved in age-related hearing loss. The mice were treated from ages 4 to 12 weeks (N ≥ 5), and auditory brainstem response (ABR) thresholds were measured at 8, 16, and 32 kHz. Treatment of the mice with a combination of <smallcaps>L</smallcaps>-methionine and valproic acid (M+V) significantly reduced the increase in the ABR threshold at 32 kHz. Treatment with any of these reagents individually produced no such effect. Microarray analyses detected 299 gene probes that were significantly up- or down-regulated in the cochleae of mice treated with M+V compared with the control vehicle-treated mice. Quantitative RT-PCR confirmed significant up-regulation of a zinc importer gene,  Hearing loss can be caused by a combination of multiple environmental and genetic factors. A variety of risk factors for progressive hearing loss, including age-related hearing loss, have been identified .Oxidative stress is widely considered a major factor associated with age-related hearing loss , whereasO-gallate (EGCG) is one of the reagents that increase zinc intracellular uptake via Zip4 induction [Zinc is an essential trace element that modulates a variety of protein functions, such as enzymatic activity, protein structure, and gene expression, and it is important for multiple physiological processes including proper functioning of the pancreas and the immune and nervous systems . The intnduction . Other nnduction , 17.DNMT1, MECP2, and HDAC8 also show multiple symptoms including hearing loss [Epigenetic regulatory pathways include DNA methyltransferases (DNMTs), histone deacetylases (HDACs), and non-coding RNAs. These pathways work in combination to change chromatin conformation and alter transcription factor accessibility to promoters, therefore altering gene expression levels . Thing loss –27.Cdh23753G>A [Fscn2326G>A alleles [in vivo experiments.Genetic variants associated with progressive hearing loss have been extensively researched in animal models as well as humans . The DBA3753G>A  and Fscn alleles , genetic alleles . Loss of alleles . AlthougZip4 can attenuate progressive hearing loss in DBA mice.Comprehensive understanding of the molecular pathways involved in hearing may lead to the prevention or treatment of hearing loss. In this study, we investigated whether epigenetic regulatory pathways could modulate the maintenance of mouse auditory function, and we demonstrated that up-regulation of in vivo treatment with epigenetic modifying reagents, male DBA mice (4 weeks old) were subcutaneously injected once daily for 8 weeks with l-methionine  and/or valproic acid , folic acid , or vitamin B12  in 0.1 M sodium bicarbonate (0.1 ml/10 g body weight). The solutions were filtered, aliquoted, and stored at -20°C. A fresh aliquot was thawed weekly and kept at 4°C. In the other experiments, DBA mice were subcutaneously injected three times weekly for 8 weeks with EGCG  dissolved to 0.05% or 0.1% final percentage in saline. The EGCG solutions were made fresh weekly, filtered, and kept at 4°C. The auditory epithelium was defined here as consisting of the organ of Corti and the spiral limbus [Inbred strains of DBA and FVB/NJ (FVB) mice, which represent a wild-type auditory system, were purchased from Clea Japan . All animals were housed in plastic cages with metal lids in a room with a 12-h light/dark cycle and 55% humidity at 23°C and had free access to food and water. For l limbus . All expABR was measured as described  with sliDnmts and Hdacs) or from the whole left cochleae of DBA mice (for other genes) at postnatal day 1 (P1), P14, 4 weeks, and 12 weeks with or without drug treatment, or from organs of adult FVB mice. Total RNA was extracted from tissues immediately after dissection using TRIZOL reagent , purified using the RNeasy mini kit , and reverse transcribed using SuperScript III (Life Technologies) with random-sequence hexamer primers . For RT-PCR, 1/100 of the reverse transcription reaction mix was used for each PCR reaction. For qRT-PCR, 1 μl of cDNA was used as the template for each reaction, which was carried out in duplicate, and the reaction products were analyzed with the Prism 7000 system  using Power SYBR Green Master Mix (Life Technologies). The standard curves for Dnmts, Hdacs, and Gapdh were generated using the cDNA generated from ovaries of adult FVB mice at 5 months of age.RNA extraction, reverse transcription, reverse transcription–PCR (RT-PCR), and qRT-PCR were performed as described , with mot-test (two-sided). To validate the microarray data, qRT-PCR was carried out to measure differences in cycle threshold values (ΔCt) between each gene and Gapdh. The standard curve for Zip4 and Gapdh was generated using mouse hepatocarcinoma Hepa1-6 cells (The Zip4/Gapdh ratio = 1 in Hepa1-6 cells) supplied by the RIKEN BioResource Center . Statistical significance was estimated by one-way ANOVA and Scheffe's test (N = 3). The primer sequences for RT-PCR and qRT-PCR are shown in For microarray gene expression analysis, the quality of RNA extracted from the whole cochlea of the left inner ear (5–6 mice in each group) was assessed with the Agilent BioAnalyzer 2100 . The RNA was subjected to a microarray assay using the GeneChip Mouse Genome 430 2.0 Array  with 45,101 probes. The expression array data were normalized using the MAS5 algorithm (Affymetrix). To correct for bias between chips, we performed quantile normalization  on all aThe dataset from the microarray experiments has been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (Accession number: GSE62173).Gene set enrichment analysis for molecular function and disease was performed using the Ingenuity Pathway Analysis software (Qiagen). The analysis was based on Fisher’s exact test. The data were adjusted for multiple tests using the Benjamini-Hochberg false discovery rate .Mice were anesthetized by xylazine-ketamine, perfused with saline, and fixed with 10% formalin. Dissected right inner ears were decalcified for 10–14 days at 4°C and paraffin embedded. Transverse 5-μm-thick sections of the inner ear were rehydrated, pretreated with 10 mM sodium citrate (pH 6.0), and blocked in phosphate-buffered saline with 0.05% Tween-20, 1% bovine serum albumin, and 5% fetal bovine serum. The specimens were incubated with rabbit antiserum against Zip4  and mouse monoclonal antibody against tubulin-βIII  at 4°C overnight. The protein signals were visualized with Alexa 488—or 568–conjugated secondary antibodies  followed by counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Images were captured with confocal microscopy . Signal specificity of Zip4 staining was determined using intestinal tissue sections from adult FVB mice .Dnmts and Hdacs in the auditory epithelia of FVB mice with normal hearing. qRT-PCR revealed that the expression levels of all three Dnmts were the highest at postnatal day (P)1 and had decreased by P14, ~4 days after the onset of hearing [Hdacs encoded in the mammalian genome were expressed in the mouse cochlea at P1, whereas at 12 weeks, some of the Hdacs, such as Hdac2, were barely detectable and appeared to be expressed at very low levels  and 12 weeks (8 weeks after treatment) of age . In untrp < 0.05, Otx1, Zip4, Calca, Pold3, Hspa1b, Serpinh1, Gabra1, and Gabrb2 to validate the expression levels by qRT-PCR, based on their known relevance to hearing [Pold3 was also selected because it is involved in DNA replication and repair [Gabra1 and Gabrb2 were considered to be representative genes for neural function, as gene set enrichment analysis based on gene annotations demonstrated that many of the genes down-regulated by M+V were categorized as having a neural function  showed a ratio of >1.5-fold for transcript levels in the M+V-treated cochleae compared with the control cochleae, and 244 probes (195 genes) showed a ratio of >1.5-fold for transcript levels in the control cochleae compared with the M+V-treated cochleae  and the vehicle control cochleae (0.15 ± 0.11) to the M+V-treated cochleae (0.65 ± 0.08) at 12 weeks . Zip4 expression, which is predominantly in the adult mouse intestine [Additional qRT-PCR analyses demonstrated that the 12 weeks . The M+Vntestine  S4 Fig)Zip4/Gapdntestine  , the ABR threshold increased by 8 ± 4 dB at 32 kHz, significantly less than untreated (23 ± 4 dB) and saline-treated (20 ± 6 dB) mice, or mice treated with EGCG at 10 mg/kg  (Zip4/Gapdh ratio was significantly higher in the EG5-treated cochleae (0.91 ± 0.56) than in the untreated (0.11 ± 0.10) or saline-treated (0.33 ± 0.18) cochleae (Zip4/Gapdh ratio in the EG10-treated cochleae (0.56 ± 0.30) did not show statistical significance compared with the other groups of cochleae. Expression levels of Otx1, Hspa1b, Gabra1, and Gabrb2 did not show significant differences after EGCG treatment  . The ABRcochleae . The Zipreatment .This study strongly suggests that epigenetic regulatory pathways can alter auditory function and identified Zip4 as a likely participant in maintaining hearing in DBA mice.Zip4 in the whole cochlea, as detected by microarray analysis and by qRT-PCR, suggests a combination of two effects: enhanced expression in the cochlear cells that express Zip4 endogenously and ectopic expression of Zip4 in the cochlear cells with undetectable expression in the uninduced state. Differences in the Zip4 signal intensity in the spiral ganglion cells among untreated, control, and M+V treated mice might be related to the injection of the solvent and not to the attenuation of progressive hearing loss. Intriguingly, cochlear tissues are rich in several enzymes that are affected by intracellular zinc, such as Cu/Zn superoxide dismutase and carbonic anhydrase [et al. [Our results support previous studies in elderly adults with hearing loss , patientnhydrase ,46. Our nhydrase . This di [et al.  and toneHspa1b [Although gene set enrichment analysis demonstrated that the expression of multiple genes related to neural function was changed by M+V , additioHspa1b , play a Hspa1b , but chaDnmt and Hdac expression in functionally mature mouse auditory epithelium suggests that epigenetic regulatory pathways are involved in auditory function. The significant prevention of progressive hearing loss by M+V, but not MET or VPA alone, strongly suggests that epigenetic modification through multiple pathways synergistically alters the transcriptional profile in cochlear cells and leads to prevention of hearing decline. The anti-oxidative effect of l-methionine, which can prevent cisplatin-induced ototoxic effects to the same extent as d-methionine [l-methionine would have resulted in increased amounts of S-adenosylmethionine, a direct methyl donor to DNA as well as histones, and thus modification of gene expression. Although Hdac inhibitors have been reported to prevent aminoglycoside-induced hair cell death in vitro [Zip4 expression and attenuation of progressive hearing loss. Although indirect effects on hearing related to changes in the physiological or metabolic status of other organs cannot be excluded, the relatively abundant expression levels of Dnmts, Hdac1, and Hdac2 in the cochlea support the idea that attenuation of progressive hearing loss by in vivo treatment with M+V is associated with direct modification of epigenetic status in cochlear cells.Detection of thionine , was insin vitro  and cispin vitro , VPA aloLow serum levels of both FA and VB12 are associated with age-related hearing loss ,52, but Zip4 expression in the cochleae or M+V treatment suggests that Zip4 induction is a major component in the attenuation of hearing loss in DBA mice. Although EGCG has multiple biological activities such as anti-angiogenic and anti-tumor activity [Zip4 induction by EGCG treatment, consistent with the effects of M+V treatment, which showed similar attenuation of progressive hearing loss in DBA mice. An alternative mechanism of EGCG treatment—other than Zip4 induction—that might have attenuated progressive hearing loss in DBA mice is its antioxidant effect. However, because treatment with MET alone failed to attenuate progressive hearing loss in our study and because stimulation of anti-oxidative activities by EGCG has been shown only in aged (not young) rat brains [Zip4 and other as-yet-unidentified target genes may help in the development of preventive therapies for hearing loss.The comparable reduction in ABR threshold increase at 32 kHz in the presence of EGCG-induced activity , we havet brains , we do nThe significant attenuation of progressive hearing loss in DBA mice treated with EGCG at 5 mg/kg, but not with 10 mg/kg, may be indicative of the effective range of EGCG doses. This finding agrees with the previous observation that dietary EGCG at 250 mg/kg does not attenuate hearing loss in C57BL/6 mice, another model of progressive hearing loss .In conclusion, this study suggests that epigenetic regulatory pathways can modify auditory function and that zinc intake in the cochlea through Zip4 mediates the maintenance of mammalian hearing. Manipulation of the expression of genes to enhance zinc intake in the cochlea should be explored as an alternative therapeutic strategy for preventing progressive hearing loss.S1 Figp < 0.05, **p < 0.01 (one-way ANOVA and Scheffe’s test).Values shown are the mean ± s.d.; *(PDF)Click here for additional data file.S2 FigABR thresholds were recorded in the left ear of each mouse before  and after treatment  with the noted drugs. Values are shown as the mean ± s.d.(PDF)Click here for additional data file.S3 FigGapdh. Otx1, Zip4, Calca, Hspa1b, Pold3, and Serpinh1 were selected from the list of genes with significantly higher transcripts and Gabra1 and Gabrb2 from the list with significantly lower transcripts in M+V-treated cochleae relative to control cochleae. Data are shown as the mean ± s.d.; *p < 0.05; **p < 0.01 .Expression levels were assessed by qRT-PCR and quantified by the ΔCt method for each gene and compared with (PDF)Click here for additional data file.S4 FigIntestinal specimens from adult FVB mice were incubated with  or without  rabbit antiserum against Zip4 (green). A and C are dark-field images of B and D. Scale bar = 50 μm.(TIF)Click here for additional data file.S5 FigABR thresholds were recorded in the left ear of each mouse before  and after  treatment with 5 mg/kg EGCG (EG5), 10 mg/kg EGCG (EG10), or saline control. Values are shown as the mean ± s.d.(PDF)Click here for additional data file.S6 FigGapdh. Otx1, Hspa1b, Gabra1, and Gabrb2 were analyzed. Data are shown as the mean ± s.d. (N = 3 mice). No statistical difference was detected by one-way ANOVA.Expression levels were assessed by qRT-PCR and quantified by the ΔCt method for each gene and compared with (PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Structural biological materials have developed heterogeneous and hierarchical architectures that are responsible for the outstanding performance to provide protection against environmental threats including static and dynamic loading. Inspired by this observation, this research aims to develop new material and structural concepts for broadband vibration mitigation. The proposed composite materials possess a two-layered heterogeneous architecture where both layers consist of high-volume platelet-shape reinforcements and low-volume matrix, similar to the well-known “brick and mortar” microstructure of biological composites. Using finite element method, we numerically demonstrated that broadband wave attenuation zones can be achieved by tailoring the geometric features of the heterogeneous architecture. We reveal that the resulting broadband attenuation zones are gained by directly superimposing the attenuation zones in each constituent layer. This mechanism is further confirmed by the investigation into the phonon dispersion relation of each layer. Importantly, the broadband wave attenuation capability will be maintained when the mineral platelet orientation is locally manipulated, yet a contrast between the mineral platelet concentrations of the two constituent layers is essential. The findings of this work will provide new opportunities to design heterogeneous composites for broadband vibration mitigation and impact resistance under mechanically challenging environmental conditions. Periodic structures and materials with spatially modulated elastic constants and densities have attracted intensive research interests due to their capabilities to manipulate the propagation of sound and heat. The introduction of inherent periodicity leads to modification of phonon dispersion relations, providing avenues to tailor group velocities12467891112151719vol.% of hard mineral aragonite imbedded within a soft organic matrix, exhibits a fracture toughness about three orders of magnitude higher than that of pure aragonite3132353637Structural biological materials have long served as a source of inspiration for developing high performance composites. Indeed, many structural biological materials such as seashells, bone, and teeth have developed sophisticated architectures enabling an unusual combination of high stiffness, high strength, fracture toughness, and energy dissipation21222324252627In this work, we investigate the elastic wave propagation in bio-inspired heterogeneous composites, aiming at addressing the bandwidth limitation and the conflict between vibration mitigation and mechanical performance of conventional periodic structures. The model system under investigation consists of two layers where each layer consists of high volume of platelet-shape reinforcements embedded in the low-volume soft matrix, which is a typical “brick and mortar” microstructure of biological structural composites see . The viba1 =  and a2 = , where l is the length of the mineral platelet, d is the thickness of the organic matrix and α is half of the lattice angle (see Vf  = 2lh/[(l+d)2·tanα], where h is the height of mineral platelet. Here we assume l = 10 μm and d = 0.2 μm, which are similar to the reported geometric parameters of nacre3940α = 30°. For these given lattice constants of the unit cell, the first Brillouin zone can be constructed accordinglyEm = 100 GPa, Poisson’s ratio vm = 0.30, and density ρm = 2950 kg/m3 for mineral platelets; Young’s modulus Eo  = 20 MPa, Poisson’s ratio vo  = 0.30, and density ρo = 1350 kg/m3 for organic matrix. The transmission spectra and phonon dispersion relations are obtained by conducting frequency domain analyses and eigenfrequency analyses, respectively. More details concerning the numerical modeling can be found in the Methods section.f = 46 ~ 96 MHz), II (f = 104 ~ 179 MHz), and III (f = 207 ~ 285 MHz), respectively. By comparing these regions with the attenuation zones of each constituent layer, we can conclude that region I and III mainly result from the attenuation zones in the lower and upper layer, respectively. However, the enhanced attenuation zones in region II are attributed to the direct superimposition of those in both lower and upper layer. If the frequency is normalized by a = 10 μm is the lattice constant and ct  = 75. 5 m/s is the transverse velocity of the matrix phase, the widths of attenuation zones in region I, II, and III are 1.05, 1.58, and 1.64, respectively. These findings indicate that broadband and enhanced wave attenuation capability can be readily achieved by designing the heterogeneous composites with two distinct constituent layers, leading to direct enhancements in vibration mitigation.f = 25.8 MHz, below the attenuation zones, the incident wave can pass through the heterogeneous composite without decay. Interestingly, when the incident frequency, f = 79.8 MHz, lie within region I, the incident wave passes through the upper layer without decay, whereas it is totally reflected by the lower layer. This observation is consistent with our finding that the attenuation zones within region I are attributed to the lower layer. When the incident frequencies, f = 127.4 MHz and f = 236.2 MHz, lie within region II and III, respectively, the incident wave is mostly reflected by the upper layer, indicating that the proposed heterogeneous composite can mitigate vibration more effectively.To qualitatively show the broadband wave attenuation capability of the proposed heterogeneous composite, we plot in To gain insight into the physical mechanisms responsible for the broadband attenuation zones of the heterogeneous composite, phonon dispersion relations need to be constructed. The proposed heterogeneous composite as a whole is not periodic; however, each constituent layer indeed is a two-dimensional periodic structure. In this regard, the phonon dispersion relation of each layer can be calculated accordingly, as shown in c, d and g, h, are believed to result from local resonances. This observation is supported by the distinctive feature that the band gaps are bounded by flat bands, indicating that zero group velocity exists and hence the energy flow is prohibited679Here the band gaps corresponding to attenuation region I, II and III are taken as examples to reveal the physical mechanisms of band gap formation. As previously predicted, the first two band gaps in each layer are due to Bragg scatteringThrough millions of years’ evolution, biological materials have optimized their shapes to handle external multidirectional loading conditions, whereby the biological materials exhibit enhanced site-specific mechanical properties2530313244We next explore the effect of local mineral platelet concentration of the proposed heterogeneous composites on the wave attenuation capability. The mineral platelet concentration in the lower layer is varied from 0.50 to 0.90, while that in upper layer remains 0.90. We have demonstrated the existence and robustness of broadband wave attenuation capability in the bio-inspired heterogeneous composites. We show that the broadband attenuation zones in the heterogeneous composites are achieved by directly superimposing the attenuation zones in each constituent layer. It should be pointed out that the constituent layers of the proposed heterogeneous composites in this work are based on the biological structural composites with high mechanical performance. We plot the stiffness and strength contour maps of the constituent layers as a function of the aspect ratio and the volume fraction of mineral platelets . Note th25303638In general, a few approaches have been proposed to improve the wave attenuation capability of composite materials, including topological optimization48We reveal that the attenuation zones in the proposed heterogeneous composites are attributed to Bragg scattering and/or local resonances. Besides dictating the band gaps of the heterogeneous composites, these physical mechanisms also contribute to the outstanding mechanical properties of structural biological compositesWe also show that the original broadband attenuation zones will be maintained and additional attenuation zones will arise when the mineral platelet orientation in the upper layer is rationally manipulated. Importantly, a contrast between mineral platelet concentrations of each constituent layer is essential to generate broadband vibration mitigation. Indeed, manipulating local characteristics of heterogeneous architectures to handle externally mechanical challenges is prevalent in structural biological composites. For example, synthetic composites with locally tunable orientation and concentration of reinforcements have been recently reported33523352In summary, we have reported broadband vibration mitigation capability in the bio-inspired heterogeneous composites that are based on the biological structural composites with high mechanical performance. Broadband wave attenuation capability is achieved by directly superimposing the attenuation zones in each constituent layer. Physical mechanisms, including Bragg scattering and local resonances responsible for the attenuation zones, have been identified by studying the phonon dispersion relation of each layer. The investigation into the effect of local characteristics of the heterogeneous composites on attenuation capability indicates that the broadband wave attenuation capability will be maintained, provided that a contrast between mineral platelet concentrations exists. The current study not only provides better understanding of the dynamic response of bio-inspired heterogeneous composites, but also opens new avenues to design optimal multifunctional composites for broadband vibration mitigation and impact resistance under mechanically challenging environmental conditions.S .


Grooming behaviour is the most common innate behaviour in animals. In rodents, it consists of sequences of movements organized in four phases, executed symmetrically on both sides of the animal and creating a syntactic chain of behavioural events. The grooming syntax can be altered by stress and novelty, as well as by several mutations and brain lesions. Grooming behaviour is known to be affected by alterations of the dopamine system, including dopamine receptor modulation, dopamine alteration in genetically modified animals, and after brain lesion. While a lot is known about the initiation and syntactic modifications of this refined sequence of movements, effects of unilateral lesion of dopamine neurons are unclear particularly regarding the symmetry of syntactic chains. In the present work we studied grooming in mice unilaterally lesioned in the medial forebrain bundle by 6-hydroxydopamine. We found a reduction in completion of grooming bouts, associated with reduction in number of transitions between grooming phases. The data also revealed the development of asymmetry in grooming behaviour, with reduced tendency to groom the contralateral side to the lesion. Symmetry was recovered following treatment with L-DOPA. Thus, the present work shows that unilateral lesion of dopamine neurons reduces self-grooming behaviour by affecting duration and numbers of events. It produces premature discontinuation of grooming chains but the sequence syntax remains correct. This deficient grooming could be considered as an intrinsic symptom of Parkinson’s disease in animal models and could present some similarities with abnormalities of motor movement sequencing seen in patients. Our study also suggests grooming analysis as an additional method to screen parkinsonism in animal models. Grooming is the most common behaviour found in mammals, birds and insects . In laboThis grooming sequence is organized in a fixed chain of behavioural events that can be considered as a syntax. This stereotyped behaviour is highly sensitive to manipulations, such as pharmacological treatments or gene mutations –7. StriaGrooming is regulated by dopamine signalling through activation of D1 and D2 receptors. D1 receptor activation plays a particular role in the grooming accomplishment. D1 receptor agonist SKF38393) increases the grooming duration and alters the rostrocaudal sequence, by reducing the face grooming and increasing the flank grooming . Moreove3 increasDue to the importance of basal ganglia, and mainly the striatum, in grooming behaviour, it is straightforward to think that diseases affecting this brain circuit, such as Parkinson’s disease (PD) can have effects on grooming. Parkinson’s disease is a progressive neurodegenerative disorder with a loss of nigrostriatal dopamine neurons leading to emergence of tremor, rigidity, akinesia, and bradykinesia. PD is most often asymmetrical, the symptoms being more pronounced in one side early in disease development and this lateralization being preserved throughout the disease . AsymmetIn the current study, we recorded grooming duration and grooming syntactic chain in mice with a unilateral 6-hydroxydopamine lesion, and compared the effects ipsilateral and contralateral to the lesion. The impact of a low dose of L-DOPA on some grooming parameters and asymmetries was also analysed.ad libitum and housed 5 per cage. All the experiments were in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals (decree 87–848). The laboratory animal facility was approved to carry out animal experiments by the Sous-Direction de la Protection Sanitaire et de l’Environnement de la Préfecture de Police . The experimental protocols were approved by the Institut du Fer à Moulin local review board. The principal investigators had a personal agreement .Thirty-seven 8-week old C57BL/6J male mice were used in two different experiments: 6 sham and 12 lesioned mice in the first experiment and 6 sham and 13 lesioned mice in the second experiment. The mice were maintained in a 12-h light/dark cycle, in stable conditions of temperature (22°C), with access to food and water Mice were anesthetized with a mixture of xylazine (10 mg/ml) and ketamine (25 mg/ml) (Centravet) and mounted in a digitalized stereotactic frame (Stoelting Europe) equipped with a mouse adaptor. 6-OHDA-HCl  was dissolved in a solution containing 0.2 g/L ascorbic acid and 9 g/L NaCl. Mice received an intraperitoneal (i.p.) injection of the selective noradrenaline reuptake inhibitor, reboxetine , 30 min prior unilateral injection  of 0.5 μMice were let on a warm plate during about 24 h after surgery to avoid hypothermia. To reduce suffering, mice received subcutaneous injections of a non-steroidal anti-inflammatory drug  just after the surgery and twice daily during the next 2 days. The weakest animals also received injections of 5% glucose  and saline  to avoid dehydration, until they reached 80% of their initial weight. Sweetened condensed milk was provided to all the lesioned animals during one week after the operation. Mice were allowed to recover for 3 weeks before behavioural evaluation and 4 weeks before drug treatment. Lesions were assessed at the end of experiments by determining the striatal levels of tyrosine hydroxylase (TH) using immunoblotting (see below). Only animals with a TH level reduction by more than 70% in the lesioned striatal area compared with the control side were included in the analyses.Mice were injected with L-DOPA 4 weeks after the lesion. L-DOPA and the peripheral DOPA decarboxylase inhibitor benserazide hydrochloride (Sigma-Aldrich) were dissolved together in physiological saline solution (9 g/L NaCl) and i.p. injected at a dose of 2.5 and 12 mg/kg, respectively, in a volume of 10 ml/kg body weight as acute treatment (one injection).Prior to testing, mice were transported from their holding room to the testing room and allowed at least 1 h for acclimation. All observations were performed between 2:00 and 7:00 pm to ensure uniformity throughout trials. Animals were individually placed in a clear cylinder  for behavioural observation. To assess spontaneous grooming, the mice were video-recorded for 2–3 h using the HomeCageScan software  by side-view and top-view web cameras (Pacific Electro-optics model PA-290 DN). Some mice were also video-recorded for 3 h after L-DOPA injection. The observation cylinder was thoroughly cleaned using 30% ethanol (vol/vol) between subjects.The videos were manually analysed as described by Kalueff et al. . During The number of vertical rears and sleeping time (“resting time”) during the observation session were also scored for each mouse. The number of clockwise and counter-clockwise rotations (360°) made by the animals were also counted during the sessions.Data were generated for the percentage of ipsilateral events for turns and grooming, compared to the total events, the percentage of complete vs. incomplete grooming bouts and for the percentage of correct vs. total transitions. A correct transition was defined following the typical cephalo-caudal progression , see details in [Sham-operated and 6-OHDA-injected mice were killed by decapitation and their heads were immediately frozen in liquid nitrogen (12 s) . The froThe membranes were immunoblotted using TH monoclonal mouse (Millipore) and actin monoclonal rabbit (Sigma) antibodies and IRDye 800CW-conjugated anti-mouse and anti-Rabbit IgG (1:5000)  as secondary antibodies. Their binding was quantified using an Odyssey–LI-COR infrared fluorescent detection system (LI-COR).post hoc tests for multiple comparisons. For two-group comparisons, two-tailed unpaired Student’s t test with Welch’s correction was used. Normality was assessed by Kolmogorov-Smirnov test with Dallal-Wilkinsons-Lieliefor P Value (Graph Prism 6).Multiple comparisons were analysed using one-way ANOVA, followed by Sidak’s Three weeks after 6-OHDA or vehicle injection in the MFB, the mice were video-recorded to study self-grooming behaviour. At the end of experiments, the TH levels were measured in the striatum of sham-operated and 6-OHDA-injected animals . Seven 6Using manual analysis, we found that grooming behaviour was decreased in unilaterally 6-OHDA-lesioned mice as compared to sham-operated animals. The number of bouts and episodes see  was reduAs compared to sham animals, we observed in 6-OHDA-lesioned mice significant behavioural asymmetries. They were characterized by a rotation behaviour towards the side of lesion  , as repoTogether, these results demonstrated that unilateral lesion of dopamine neurons induced changes in self-grooming behaviour, not only affecting duration and numbers of events, but also breaking the grooming chains as evidenced by the reduced number of transitions and percentage of complete grooming chains. However, the syntax of grooming events appeared to be preserved since the percentage of correct transitions was not affected by the lesion. The unilateral disruption of nigrostriatal pathway altered the characteristic symmetry of this behaviour by producing reduced contralateral grooming.To test the possibility that L-DOPA at low dose (2.5 mg/kg) could have a positive effect on the grooming changes and asymmetries developed after surgery, a new cohort of animals was used. Before performing the surgery, animals were recorded for 3 h in the same conditions used in the previous experiment to analyse the basal level of grooming. Mice were video-recorded again 3 weeks after lesions to analyse asymmetries in rotation and grooming as well as other grooming parameters. Four weeks after surgery both groups of animals  were treated once with 2.5 mg/kg of L-DOPA (associated with 12 mg/kg of benserazide), a dose that produced few abnormal involuntary movements characteristic of LID. To avoid that LID interferes with grooming analysis, grooming and rotation behaviours were analysed between 1.5 and 3 h after L-DOPA injection.Asymmetries in turning and grooming behaviours developed after the lesion, as compared to the analysis prior to surgery . L-DOPA L-DOPA-treated animals showed a general increase in the total duration of grooming , compareIn rodents grooming is a natural behaviour characterized by a complex pattern of actions, forming a syntax chain ,24 GroomAlthough major findings about grooming features resulted from studies of the effects of lesions in various brain regions, to our knowledge, none reported changes in grooming syntax following unilateral 6-OHDA-induced lesions as a model for hemiparkinsonism. Moreover, the effects of this lesion on grooming asymmetries were not investigated. One study in 6-OHDA-lesioned rats reported intense grooming directed toward the contralateral side to the lesion following treatments with various dopamine agonists but did not describe grooming deficits in untreated animals . Our stuDespite the general reduction in grooming activity, no change was found in the single bout duration, suggesting that each grooming phase was lasting longer and syntactic grooming chain was more frequently interrupted before completion. This is consistent with the reduction in number of transitions and rate of complete grooming observed in 6-OHDA lesioned animals. These results are also in agreement with those found after bilateral lesion of nigrostriatal neurons . In thisWe also found a development of ipsilateral preference in grooming syntax execution, resulting probably from a deficit in grooming behaviour on the contralateral side to the lesion. Behavioural asymmetries after lesion were already found to occur for turning and scanning behaviours, but not for grooming , 26. AsyL-DOPA administration also had effects on other grooming parameters. The main effect was a general increase in total grooming duration, an effect that could be related to the increase of general activity stimulated by L-DOPA . In addiSimultaneous activation of both D1 and D2 receptors at postsynaptic levels can explain the hyperactivity  and chanThe fact that self-grooming is an innate motor behaviour and the grooming syntax pattern is conserved among mammals suggests that the components of neural circuits supporting this behaviour have been optimized during evolution. In addition, it is possible that the brain structures initially specialized to carry out grooming, were later diverted to other kinds of elaborated behaviours such as speech or complex sequence learning. For example, mutation of FoxP2 which is responsible for speech deficits in human produces impairment in motor learning in mice . LanguagOur findings indicate that analysis of self-grooming activity can be a novel behavioural test to evaluate parkinsonian behaviour in animals with a lesion of dopamine neurons. In addition they demonstrate that a low dose of L-DOPA is able to rescue alterations of grooming symmetry. Finally, they show that sequence completion is affected in unilaterally lesioned animals as in those in which the lesion of dopamine neurons was bilateral  or those


Cdh23 and Fscn2, are characterized by early onset hearing loss at as early as three-weeks of age  and are an animal model for progressive hearing loss research. Recently, it has been reported that epigenetic regulatory pathways likely play an important role in hearing loss  as methyl donor and valproic acid (VPA) as a pan-histone deacetylases (Hdac) inhibitor, showed a significant attenuation of progressive hearing loss by measuring their auditory brainstem response (ABR) thresholds . Here we present genome wide expression profiling of the DBA/2J mice cochleae, with and without treatment of MET and VPA, to identify the genes involved in the reduction of progressive hearing loss. The raw and normalized data were deposited in NCBI's Gene Expression Omnibus (GEO ID: GSE62173) for ease of reproducibility and reanalysis.DBA/2J mice, which have homozygous mutations in  Mice were housed in plastic cages with metallic lid in a room with a 12-h light/dark cycle and 55% humidity at 23 °C and had free access to food and water. Male DBA/2J mice at 4-weeks postnatal age were used as non-treatment young controls . In vitr2.2Total RNA was extracted from the whole left cochleae of mice using TRIzol reagent  and further purified using the RNeasy Mini Kit  according to the manufacturer's instruction. The quality of the RNA was assessed using Agilent 2100 Bioanalyzer . We used only the high quality RNA (RIN score > 7), derived from 5 to 6 mice in each group, for microarray experiments . For mic2.3Gene expression array data were normalized using the MAS5 algorithm (Affymetrix). The intensities were converted to a logarithmic scale (base 2). To correct for bias between chips, we then performed quantile normalization 2.4Otx1, Slc39a4 , Calca, Pold3, Hspa1b, Serpinh1, Gabra1 and Gabra2) to validate the expression levels by qRT-PCR Among the genes that were highly differentially expressed between the MET + VPA and vehicle control group in microarray data, we selected 8 genes (2.5t-test. After selecting only those probes assigned a present (P) flag for all samples in at least one group for the statistical tests, 24,787 probes remained. Genes were considered differentially expressed with a p-value < 0.05 and a fold change ≥ 1.5.Using these criteria, 55 probes (49 genes) and 244 probes (195 genes) were up- and down-regulated in the MET + VPA group, respectively  from the microarray data. In addition to Scl39a4, other genes differentially expressed in the presence of the MET and VPA may contribute to the observed attenuation effect on progressive hearing loss. We believe that this expression profiling dataset would be valuable for further investigating and understanding of the molecular mechanism in hearing loss.We present a dataset of microarray expression profiling of the DBA/2J mice cochleae treated with and without an epigenetic modifying reagent to investigate the genes involved in the reduction of progressive hearing loss. In our previous report The authors declare no conflict of interests.