Rupture of atherosclerotic plaque can cause acute malignant heart and cerebrovascular events, such as acute coronary heart disease, stroke and so on, which seriously threaten the safety of human life and property. Therefore, the early diagnosis and inhibition of atherosclerotic plaque progress still be a vital task.3@SiO2-mSiO2, CMSN)-based nanomedicines (NMs) (Ru(bpy)3@SiO2-mSiO2@SRT1720@AntiCD36, CMSN@SRT@Anti) for accurate diagnosis and treatment of atherosclerosis (AS). In vitro cell experiments showed that both RAW264.7 and oxidized low density lipoprotein (ox-LDL)-stimulated RAW264.7 cells could significantly uptake CMSN@SRT@Anti. Conversely, little fluorescence signal could be observed in CMSN@SRT group, showing the excellent targeting ability of CMSN@SRT@Anti to Class II scavenger receptor, CD36 on macrophage. Additionally, such fluorescence signal was significantly stronger in ox-LDL-stimulated RAW264.7 cells, which might benefit from the upregulated expression of CD36 on macrophages after ox-LDL treatment. For another, compared with free SRT1720, CMSN@SRT@Anti had a better and more significant effect on the inhibition of macrophage foaming process, which indicated that drug-carrying mesoporous silicon with targeting ability could enhance the efficacy of SRT1720. Animal experimental results showed that after the abdominal injection of CMSN@SRT@Anti, the aortic lesions of ApoE-/-mice could be observed with obvious and persistent fluorescence signals. After 4 weeks post-treatment, the serum total cholesterol, aortic plaque status and area were significantly improved in the mouse, and the effect was better than that in the free SRT1720 group or the CMSN@SRT group.In this study, we presented the development of composite mesoporous silica nanoparticle (Ru(bpy)The designed CMSN@SRT@Anti with excellent biocompatibility, high-performance and superior atherosclerosis-targeting ability has great potential for accurate identification and targeted therapy of atherosclerotic diseases.The online version contains supplementary material available at 10.1186/s12951-021-00962-w. Cardiovascular disease (CVD) is a widely recognized human killer, and as a representative CVD, coronary heart disease (CHD) has a very high mortality rate, accounting for more than half of the deaths of CVD cases worldwide every year , 2. AS i3O4 to obtain near-infrared fluorescence imaging/MRI nanoprobe, which could locate AS plaques by imaging and moderate the progression of AS [NMs can make use of the characteristics of disease composition to cleverly identify and interfere with the outcome of disease. The well designed NMs have been widely applied in various diseases such as the diagnosis and therapy of tumor, cardiovascular disease , the inhon of AS . In the on of AS . In the on of AS .3Cl2 and the targeted binding ability of AntiCD36 to CD36 overexpressed on the surface of macrophage, we could track the AS plaque and the inhibition effect of AS progress specifically both in vitro and in vivo.The above successful examples for the molecular diagnosis and treatment of AS stimulates us to explore new probe to improve the theranostic functions. In this study, we selected CD36 as a target for the molecular diagnosis and treatment of AS. Studies have shown that CD36, which is expressed on the surface of macrophage, is a key regulatory point for the formation and development of AS. It can mediate the conversion of macrophage into foam cell after phagocytosis of ox-LDL , 20. Ant3@SiO2 and CMSN were observed by Transmission Electron Microscopy (TEM). As shown in Fig. 3@SiO2 with an average size of 28.6 ± 2.5 nm and mesoporous structure for CMSN with an average size of 61.4 ± 7.9 nm were displayed with well dispersion. Fourier transform infrared spectra (FT-IR), ultraviolet–visible (UV–vis) absorption spectra and zeta potential determination were employed to monitor the whole assembly procedure of CMSN@Anti . All of these characterizations indicated that AntiCD36 had been successfully attached to the surface of CMSN.The morphology of prepared Ru(bpy)Antibody . The zet3Cl2, CMSN presented better fluorescence stability under 365 nm UV exposure . Therefore, a concentration of CMSN@SRT@Anti below 40 μg/mL was chosen for the following cell imaging and the evaluation of inhibition effect of macrophage foaming.Macrophages, as the core cells of AS, are important components involved in the occurrence and development of AS. In this study, mouse macrophage cell line, RAW264.7 was used as the experimental object. Prior to study the targeted fluorescence imaging and macrophage foaming inhibition of CMSN@SRT@Anti, thiazole blue tetrazole bromination (MTT) assay was first introduced to evaluate their cytotoxicity. As shown in Additional file P < 0.001 vs. unstimulated cells), which might benefit from the upregulated expression of CD36 on macrophages after ox-LDL treatment [P < 0.05 ox-LDL stimulation group vs. control group) . Compared with MG, CMSN@SRT at 16 μg/mL or 24 μg/mL could decrease the cell TC value (**P < 0.01), but there was no significant difference in the effect between the two groups (P > 0.05) , but there was no significant difference in the effect between the two groups (P > 0.05)  in cells could reflect the degree of lipid deposition to indicate the degree of cell foaming. Compared with the model group (MG), either CMSN, CMSN@Anti or AntiCD36 could reduce the cell TC value little , except a transient increase in Aspartate aminotransferase (AST), which returned to normal level after 7 d. The body weight of the mice showed a slight weight loss on the first day after CMSN@SRT@Anti injection and a gradual increase thereafter, with a growth curve similar to that of the control group .In order to select the best imaging time point of nanoparticles in vivo, CMSN@SRT@Anti were intraperitoneally injected into AS model mice. The fluorescence imagings of aorta at different time points were collected and shown in Fig. P < 0.01). Notably, CMSN@SRT@Anti reduced the area of aorta red stain more effectively than CMSN@SRT (**P < 0.01) or SRT1720 (*P < 0.05), indicating CMSN@SRT@Anti had the best anti-AS effect in vivo. We also carried out quantitative analysis on serum TC values of mice in each group after different treatments. Learnt from Fig. P < 0.01 vs. Vehicle group). Similarly, CMSN@SRT@Anti treatment resulted in a significant decrease in serum TC content than CMSN@SRT or SRT1720 (**P < 0.01).To further visually display the anti-atherosclerosis effect of CMSN@SRT@Anti, the aortas of mice with the intervention were dissected and stained with Oil Red O and the ORO-positive area were quantitatively analyzed Fig. a. This iHistological cross-sections were stained with H&E to further confirm such inhibition effect Fig. c. Compar3Cl2 doped mesoporous silicon was chosen as the nanocarrier in this work. Mesoporous silicon (MSN) has been widely used in molecular diagnosis and treatment of various diseases and has been recognized as a General Recognized as Safe (GRAS) by the U.S. Food and Drug Administration (FDA) for its non-toxic, stable and biocompatible properties. Hollow MSN is an ideal drug-loading nanoplatform with low density, large specific surface area, strong surface plasticity and large drug loading capacity [3Cl2 is widely used in fluorescence labeling due to its good photobleaching resistance and wide detection range. Specifically, when Ru(bpy)3Cl2 was sealed in silicon spheres, its fluorescence stability can be significantly improved [2 was further improved, and the surface charge was changed from − 28.1 ± 0.5 mV to 24.5 ± 0.4 mV, which was consistent with the characterization of mSiO2 amination by Dai Y. et al. [2 was successfully prepared. Using Rhodamine 6G as the reference material, the fluorescence quantum yield of CMSN was as high as 52%, indicating that CMSN had a good application prospect as a fluorescent agent. Furthermore, their fluorescence signal still remained at a high level even under the irradiation of 365 nm UV lamp for 1 h, and a stable fluorescence signal under a wide range of pH (1–10) environments, indicating their good anti-photobleaching ability and stability to pH changes, which was consistent with the description of Ru(bpy)3@SiO2 fluorescence stability by Ho Ta et al. [3Cl2 was sealed in the silicon sphere, which could effectively block Ru(bpy)3Cl2 agglomeration. No significant change in hydrated particle size of CMSN@Anti for 9 days standing at room temperature further showed their excellent colloid stability, which was necessary for the biomedical applications.At present, NMs have been widely used in the scientific research of various diseases, such as cancer, metabolism-related diseases, etc. . Howevercapacity –31. On timproved . Thus, bimproved , CMSN as. et al. , indicata et al. . In this2 to target CD36 for the track and treatment of AS plaque. The fabricated CMSN@Anti was analyzed by Zeta potential apparatus, UV–vis and FT-IR. The existence of specific targeting molecule, AntiCD36 not only favors the track of AS plaque, but also help to improve the drug delivery efficiency and therapy effect eventually. Nicotinamide adenine dinucleotide (NAD +) dependent class III histone deacetylase (SIRT1) has been dubbed as the “longevity gene” [vitro and in vivo experiments. In vitro experiment results showed that CMSN@SRT@Anti could effectively reduce TC content in RAW264.7 cells. In addition, the TC lowering effect of CMSN@SRT@Anti was stronger than that of SRT1720 alone, indicating that CMSN@SRT@Anti with targeting ability can enhance the therapeutic effect of SRT1720 on AS [vitro, the relatively static culture environment in the cell dish was conducive to the phagocytosis of macrophages, while in vivo, the nanomaterials were constantly flowing in the blood circulation, and the accumulation of CMSN@SRT without targeting ability were less in the lesions of AS, which was not conducive to the treatment of AS. In addition, due to the low dose of AntiCD36 contained in CMSN@SRT@Anti, the NMs failed to show the competitive inhibitory effect of AntiCD36 on the treatment of AS [In recent years, a large number of studies have shown that macrophages play a decisive role in the pathological process of AS. Early ideas suggested that unstable plaques were characterized by a large necrotic center with a large infiltration of macrophages , 35. Howty gene” , which cty gene” . In addity gene” . In thisty gene” . Nanomat20 on AS . Similarnt of AS .vivo is an important index to evaluate the safety of NMs. Studies have shown that the distribution and metabolism of NMs in organisms are related to the size of NMs and the characteristics of viscera. NMs with particle size less than 5.5 nm are usually metabolized through the urinary system, while NMs with particle size greater than 15 nm are not easy to pass through the glomerulus and are eventually excreted to the cholecyst through the liver [The metabolism of NMs in he liver . The parhe liver , 20. In However, there are some limitations in this study. For example, the excitation visible fluorescent dye selected in this project has poor penetration, and its penetration in vivo is far less strong than that of near-infrared fluorescent dye, resulting in an increase in experimental consumables and labor costs for NMS imaging and metabolism detection in vivo.In the current study, we used mesoporous silicon as a nanoplatform to synthesize NMs for the precise diagnosis and treatment of atherosclerotic lesions. AntiCD36 was chosen as target molecule to recognize macrophages and SRT1720 loaded in mesoporous acted as the drug to inhibit the progress of AS plaque. It should be mentioned that such fabrication of functional NMs not only realized the tracking of AS lesions, but also enhanced the therapy efficacy of free SRT1720, which provided a new avenue for the applications of nanomaterials in the diagnosis and therapy of AS.3@SiO2. Typically, 3.5 mL of Ru(bpy)3Cl2 solution (10 mM in H2O) was added to a mixture of 35 mL cyclohexane, TritonX-100 and hexanol with a ratio of 4:1:1. The obtained mixture was maintained at room temperature for 30 min under magnetic stirring. Then, 1 mL mixed solution of TEOS and NH3.H2O with a ratio of 1:1 was added and was continued stirred for 24 h. At the end of the reaction, the mixed solution was centrifuged  to obtain a light red precipitate, named Ru(bpy)3@SiO2. The product was washed with ethanol for 3 times, and then dried in a lyophilizer and stored at 4 °C away from light.The fabrication of the CMSN@SRT@Anti began with the preparation of Ru(bpy)2 layer was used for drug delivery in the current study. Briefly, 25 mL N-Hexadecyltrimethylammonium chloride (CTAC) aqueous solution (25% wt) and 250 μL triethylamine (TEA) were added into 35 mL Ru(bpy)3@SiO2 solution (3 mg/mL). After mixing evenly, 20 mL tetraethyl orthosilicate (TEOS)  was added. The mixture was stirred at 60 °C (200 rpm) for 24 h without light. After the reaction, the obtained product was demoulded by NaCl-methanol solution (1% wt) . The obtained CMSN was washed, dried and finally preserved at 4 °C in isolation from sunlight.The covering of mSiOThe CMSN@SRT@Anti was formed in three steps. Firstly, the amination of CMSN was performed. 20 μL (3-Aminopropyl) triethoxysilane (APTES) was dropped in 3 mL CMSN solution (10 mg/mL in anhydrous toluene) slowly, and they were stirred at 60 °C for 5 h under nitrogen protection. The precipitate was washed by anhydrous ethanol for three times and dried for later use. Secondly, SRT1720 was loaded. Briefly, 30 μL SRT1720 solution (50 mM in Dimethyl sulfoxide) were incubated with 470 μL amino modified CMSN (1.0 mg/mL) overnight in a thermostatic oscillator . After the reaction, the centrifugal precipitate was washed with PBS for three times to remove the unloaded drugs. Finally, covalent binding of CMSN@SRT with AntiCD36 was performed. Briefly, 4 μg AntiCD36 was dissolved in 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution (2 mg/mL in 500 μL PBS) for 15 min for carboxyl group activation. Then, it was mixed with 500 μL solution containing 1 mg CMSN@SRT and 1 mg N-hydroxysuccinimide (NHS) for 2 h incubation at 37 °C. After centrifugation and washing, the precipitates were dispersed in 1 mL PBS and stored at 4 °C before use. The CMSN@Anti were prepared following the same procedures as described above, except without SRT1720 loading.3@SiO2 and CMSN were characterized by TEM . The UV–vis absorption spectra and the fluorescence spectra were recorded by an ultra-micro spectrophotometer  and a fluorescence spectrophotometer , respectively. FT-IR spectra were detected by Fourier transform infrared spectrometer . The surface potential and hydrated particle size were detected by Zeta potentiometer .Ru(bpy)2 nanoparticles were mixed with different concentrations of SRT1720  and the mixture was shaken overnight by an oscillator at 37 °C. The obtained solution was ultracentrifuged using a 100 kDa ultrafiltration cube and the unloaded SRT1720 amount in the filtrate was determined by UV–vis absorbance at 246 nm. Then, the AntiCD36 was conjugated with CMSN@SRT and the lost SRT1720 in this process was also determined to calculate the final SRT1720 loading amount in CMSN@SRT@Anti. The encapsulation efficiency (EE) and loading efficiency (LE) of CMSN@Anti for SRT1720 were calculated using the following equations:In order to evaluate the drug loading and drug release of CMSN@SRT@Anti in vitro, the standard curve of SRT1720 was firstly determined with the UV–vis absorbance intensity of SRT1720 at 246 nm as the ordinate and the corresponding concentrations (0.01–5 mM) as the abscissa. Then, 0.5 mg CMSN-NHvitro release is as follows. 0.5 mL CMSN@SRT@Anti (0.25 mg/mL) was transferred into a dialysis bag (MWCO = 100 KDa) and placed in tubes containing PBS with pH of 6.5, 7.4, or 9 respectively. They were rotated at 37 °C with a speed of 200 rpm for 48 h. 2 μL liquid in the tubes was picked up at scheduled time points, and their UV–vis absorbance values were measured using an ultra-micro spectrophotometer. The PBS buffer volume in the test tube remains constant.The method of in 2 incubator at 37 °C. For in vitro AS simulation, RAW264.7 cell were pretreated with 60 μg/mL ox-LDL .RAW264.7 (mouse macrophage cell line) and NIH-3T3 (mouse embryonic fibroblast cell line) were purchased from the Cell Bank of the Chinese Academy of Sciences . They were incubated with DMEM  supplemented with 10% fetal bovine serum  and 1% Penicillin–Streptomycin . The cells were maintained in a 5% COThe cytotoxic effect of CMSN@SRT@Anti on RAW264.7 or NIH-3T3 was evaluated by the MTT method. After the incubation of RAW264.7 or NIH-3T3 with different concentrations of nanomaterials in 96-well plates (n = 6) for 24 h (0–40 μg/mL), the media was removed. After washing with PBS, 100 μL 1 mg/mL MTT  was added to each well. After further culture for 4 h, the MTT solution was discarded gently and 100 μL of Dimethyl sulfoxide (DMSO)  was added to dissolve the formazan crystals. Finally, the absorbance of each well at 490 nm was measured with a microplate analyzer .For in vivo biocompatibility evaluation, hematological and biochemical analyses of mouse with intraperitoneal injection of CMSN@SRT@Anti were carried out. The Kunming mice were treated with CMSN@SRT@Anti or an equivalent volume of PBS at a dose of 170 mg/kg/d every other day. Eyeball blood samples were taken at 1 day, 7 day and 21 day after nanomaterials injection as well as 21 day after PBS injection. The collected blood was used for blood biochemical detection (n = 3) and routine blood detection (n = 3). Hematoxylin–eosin (H&E) staining was performed for the main organs of the mice after different treatment and observed under an optical microscope.To further study the metabolism of CMSN@SRT@Anti, the Kunming mice were injected intraperitoneally with the nanomaterials (170 mg/kg). The mice were sacrificed at scheduled time points . Fluorescence accumulation in liver and cholecyst were semi-quantified by fluorescence imaging software .To show the specific targeting ability of our fabricated CMSN@SRT@Anti to macrophages, RAW264.7, NIH-3T3, RAW264.7 pre-treated with 60 μg/mL ox-LDL or NIH-3T3 pre-treated with 60 μg/mL ox-LDL were first inoculated in 6-well plates, respectively (n = 3). Then, the cells were then incubated with CMSN@SRT@Anti or CMSN@SRT with a concentration of 16 μg/mL for 2 h at 37 °C. After fixed with 4% paraformaldehyde at room temperature for 15 min, the cells were washed twice with PBS and the nuclei were stained with DAPI. The cellular fluorescence imaging was observed under an inverted fluorescence microscope .RAW264.7 cells were evenly inoculated into 6-well plates and divided into control group and ox-LDL-treated group (60 μg/mL) (n = 3). After different treatment, cells were harvested and total protein from cells was extracted by using Radio Immunoprecipitation Assay (RIPA) lysis buffer. After separated with sodium dodecyl sulfate-poly- acrylamide gel electrophoresis (SDS-PAGE) gels, samples were then transferred to nitrocellulose (NC) membrane. The membrane was blocked with 5% nonfat dried milk diluted in Tris buffered saline Tween (TBST) for 1 h at room temperature and then incubated with primary antibodies against β-actin and CD36 overnight at 4 ℃. After washed with TBST, the membranes were further incubated with corresponding secondary antibodies for 1 h at room temperature. Finally, the protein bands were detected on Tanon 5200 chemiluminescence imaging analysis system  using efficient chemiluminescence (ECL) reagent and analyzed with the ImageJ software.To further evaluate the therapy effect of CMSN@SRT@Anti in vitro, macrophage cells were treated with different groups: blank control group , Model group , CMSN group (RAW264.7 was incubated with ox-LDL and CMSN), CMSN@Anti group (RAW264.7 was incubated with ox-LDL and CMSN@Anti), CMSN@SRT group (RAW264.7 was incubated with ox-LDL and CMSN@SRT), CMSN@SRT@Anti group (RAW264.7 was incubated with ox-LDL and CMSN@SRT@Anti), free AntiCD36 group (RAW264.7 was incubated with ox-LDL and AntiCD36), and free SRT1720 group (RAW264.7 was incubated with ox-LDL and SRT1720). The concentrations of ox-LDL in all groups were 60 μg/mL. After different treatments, the TC contents in cells were quantitatively analyzed using the TC assay kit  (n = 3).In order to more intuitively demonstrate the inhibitory effect of different treatments on the RAW264.7 cell foaming, oil red staining was carried out to instead TC value determination. ImageJ software was used to semi-quantitatively analyze the red stained area (n = 3).The cell oil red O staining method was referred to the instruction. In brief, the treated cells were fixed with 4% paraformaldehyde  at room temperature for 15 min. After rinsed with PBS twice, the oil red working solution was added to stain for 40 min in the dark. Then, the cells were rinsed with distilled water for three times and observed under a fluorescent inverted microscope. The configuration of the oil red O working liquid was prepared as follows. 6 mL 0.5% saturated oil red O was mixed with 4 mL distilled water, and purified by filtering through a filter paper in the dark.The purchased six week-old male ApoE-/-mice , initially fed a normal diet feeding 1 week to adapt to the new environment. Then, mice continued to be fed with the normal diet as a negative control group  and a  for 12 weeks to establish AS model. Composition of HFD is 40% Fat and 1.25% Cholesterol. Kunming male mice  were used to study the potential toxicity and metabolism of our prepared nanomaterials. All animal procedures followed the regulations of the Chinese Physiological Society for the Management of Laboratory Animals. The experimental scheme was in line with the ethical requirements of the Animal Care Committee of Xuzhou Medical University (202008A077).In order to select the best imaging time point of nanoparticles in vivo, 5 male AS-model mice (HFD for 16 w) with similar weight and status were used. 4 of them were treated with intraperitoneal injection with CMSN@SRT@Anti (170 mg/kg) respectively. The mice were sacrificed at different time points , and the isolated aortas were collected. Then, the arteries were washed with saline three times and visualized using a small animal fluorescence imaging system.To further compare the specific targeting ability of CMSN@SRT@Anti and CMSN@SRT, the AS-model mice were intraperitoneal injection with PBS, CMSN@SRT@Anti or CMSN@SRT with an amount of 170 mg/kg (n = 3), respectively. Four hours later, the isolated aortas were washed with saline and placed in a small animal fluorescence imaging system. The signal intensities of whole arteries were quantified using Tanon Image Gel software .To evaluate the therapy effect of AS by CMSN@SRT@Anti in vivo, ApoE−/−mice (HFD for 12 w) were divided into five groups(n = 4): the LFD + PBS group (LFD), the HFD + PBS group (Vehicle), the HFD + CMSN@SRT@Anti, the HFD + CMSN@SRT, and the HFD + free SRT1720 group (SRT1720). AS-model mice fed with HFD were intraperitoneal injected with nanomaterials (170 mg/kg/d) or SRT1720 (50 mg/kg/d)  or the sThe Oil Red O staining procedure to observe the plaque of lipid deposits was as follows. The aorta was dissected, and the adventitial fat was removed after being opened longitudinally with the luminal surface toward the outside. After fixed in 4% paraformaldehyde at room temperature for 2 h, the aorta was placed in 0.3% Oil Red O working solution  at 45 ℃ for 1 h in the dark condition, and then rinsed with 85% isopropanol for 3 times. The ImageJ quantitatively analyzed the aorta .t test was used for comparison between the two groups. According to the test level α = 0.05, P < 0.05 was considered to be statistically significant.All the analyses involved in this project were performed by SPSS 22.0 software. Data were expressed as mean ± standard deviation. Shapiro–Wilk test was used to determine the normality of the data distribution. The multi-group comparisons were made with a one-way ANOVA analysis, followed by Dunnett’s post hoc test. The Student’s Additional file 1:Fig. S1 a. The fluorescence intensity values of Ru(bpy)3Cl2 and CMSN at various time points under the UV lamp. b The hydrated particle size of CMSN@Anti. . Fig. S2 a. The UV-vis absorption spectra of SRT1720 at different concentrations. b The corresponding linear regression equation. Fig. S3. Toxicity test of CMSN@SRT@Anti to RAW264.7 or NIH-3T3 cells . Fig. S4. Representative blots and quantification of protein levels of CD36 to the β-actin levels before and after ox-LDL treatment. (*p < 0.05). Fig. S5. Blood index analysis of Kunming mice before and after CMSN@SRT@Anti intervention. ALT (P=0.624), TP (P=0.972), ALB (P=0.504), CREA (P=0.577), UREA (P=0.849), WBC (P=0.984), RBC (P=0.664), HGB (P=0.775), HCT (P=0.707), MCH (P=0.965), MCV (P=0.963), P values are all greater than 0.05. Compared with AST 0d group, P(AST 1d)= 0.023<0.05, P (AST 7d)= 0.448>0.05, P (AST 21d)= 0.313>0.05. Fig. S6. Toxicity test of CMSN@SRT@Anti on Kunming mice. a The body weight of Kunming mice injected intraperitoneally with NMs or PBS changed with time. b Pathological section of major organs in Kunming mice . Table S1. EE and LE of CMSN@SRT@Anti at different concentrations of SRT1720. Table S2. Relative fluorescence intensity of liver and cholecyst of Kunming mice at each time point after intraperitoneal injection of CMSN@SRT@Anti determined by Tanon Image softerware.


Sensory input has profound effects on neuronal organization and sensory maps in the brain. The mechanisms regulating plasticity of the auditory pathway have been revealed by examining the consequences of altered auditory input during both developmental critical periods—when plasticity facilitates the optimization of neural circuits in concert with the external environment—and in adulthood—when hearing loss is linked to the generation of tinnitus. In this review, we summarize research identifying the molecular, cellular, and circuit-level mechanisms regulating neuronal organization and tonotopic map plasticity during developmental critical periods and in adulthood. These mechanisms are shared in both the juvenile and adult brain and along the length of the auditory pathway, where they serve to regulate disinhibitory networks, synaptic structure and function, as well as structural barriers to plasticity. Regulation of plasticity also involves both neuromodulatory circuits, which link plasticity with learning and attention, as well as ascending and descending auditory circuits, which link the auditory cortex and lower structures. Further work identifying the interplay of molecular and cellular mechanisms associating hearing loss-induced plasticity with tinnitus will continue to advance our understanding of this disorder and lead to new approaches to its treatment. •During CPs, brain plasticity is enhanced and sensitive to acoustic experience.•Enhanced plasticity can be reinstated in the adult brain following hearing loss.•Molecular, cellular, and circuit-level mechanisms regulate CP and adult plasticity.•Plasticity resulting from hearing loss may contribute to the emergence of tinnitus.•Modifying plasticity in the adult brain may offer new treatments for tinnitus. In the auditory domain, frequency receptive fields of neurons form an orderly tonotopic map. Tonotopy is preserved along the auditory neuraxis and reflects the topographically organized projections from the cochlea. Organization of the tonotopic map is genetically predetermined and laid out coarsely during prenatal development. It is subsequently refined in an activity-dependent manner, first by spontaneous activity and, following hearing onset, by evoked activity . The cru2Critical periods (CPs) are defined epochs of early postnatal development, when structural and functional neural circuits are shaped by passive experience with the external environment . These wPlasticity of the cortical tonotopic map is the chief model of CP plasticity in the auditory system. The tonotopic map is established during a CP for frequency tuning, when passive acoustic experiences influence map organization across species . In rodeInterestingly, the timing of CPs is itself plastic and can be influenced by the sensory environment . UnstrucThe absence of early sensory experience is expected to have the most profound effects on auditory cortical representations. Rodent studies indicate that complete sensorineural hearing loss during development disrupts synaptic inhibition and increases excitability throughout the central auditory pathway, resulting in broadened frequency tuning  exposure to moderate-intensity tones is associated with reduced neural excitability in the cat auditory cortex . This reLater in life may also be a period during which sensory representations are unusually plastic. Noisy or absent sensory inputs caused by natural age-related degeneration of the auditory periphery may contribute to dysregulated plasticity in the aged auditory cortex . Aging i4The findings above Section  demonstr4.1The onset of sensory experience triggers the maturation of inhibition , which mA receptors -positive cell densities  and are distinct from the two other commonly studied interneuron classes expressing PV or SST (3AR-positive interneurons expressing neuron-derived neurotrophic factor (NDNF) also play a role in plasticity that underlies adult associative learning  modulates plasticity in adult primary visual cortex by inhibiting SST interneurons  and myelin, each of which may act as structural barriers to plasticity by prohibiting the formation of new synapses . PNNs arn cortex . In vivohibition  and a rehibition . PV intehibition .Less clear is the role of sensory deprivation in adulthood on the dynamic regulation of PNNs. In the adult auditory cortex, a marked cell type- and layer-dependent decrease in PNNs in the mouse primary auditory cortex occurs following noise-induced hearing loss . HoweverThe maturation of myelin is one of the last steps of development, with central auditory myelination in both humans and rodents beginning with the onset of hearing and continuing until the age of sexual maturity . Myelin 4.3de novo formation of synapses as a mechanism underlying restoration of activity in areas of deprived peripheral input. In seminal studies, focal binocular retinal lesions resulted in the deprived area of the primary visual cortex, termed the lesion projection zone, to initially become quiescent. Subsequently, however, the area regained activity and responded to loci neighboring the lesion , which has two main structural and functional divisions: the ventral and the dorsal CN. The dorsal CN relays directly to the inferior colliculus (IC), whereas the ventral CN first relays to the superior olivary complex (SOC), thought to be specifically involved in the binaural integration of acoustic input and sound localization. Projections from the SOC again lead primarily to the IC. From the IC, auditory signals are relayed to the medial geniculate body (MGB) in the thalamus, and, from there, to the primary auditory cortex. Subcortical auditory relays are thought to play an important role in the binaural processing of sound, as well as the integration with non-auditory information  increase in transmitted firing rates and conduction speed . In the 5.3During auditory CPs, neurons of the auditory brainstem undergo dramatic morphological rearrangements that are dependent on auditory input  studies have found no evidence of cortical tonotopic reorganization when examining subjects with tinnitus but normal or near-normal hearing thresholds . A recenLack of cortical reorganization in humans with tinnitus is consistent with brain imaging studies in the visual and somatosensory domains. In the visual domain, macular degeneration is a condition which, like high-frequency hearing loss, deprives a limited part of the cortical sheet of its input. fMRI studies have shown that, even after years of visual input deprivation due to macular degeneration, the relevant cortical regions remain non-responsive e.g., , suggestAs another strategy to disentangle the effects of hearing loss and tinnitus, new lines of research have begun to examine the contribution of presumably non-traumatic sound experience—potentially causing hidden forms of hearing loss—to changes in cortical responses  programs. In humans, there appear to be not only individual differences in the susceptibility to tinnitus, which may result from intrinsic genetic differences , but als


Hearing loss is a major public health challenge. Audiology services need to utilise a range of rehabilitative services and maximise innovative practice afforded by technology to actively promote personalized, participatory, preventative and predictive care if they are to cope with the social and economic burden placed on the population by the rapidly rising prevalence of hearing loss. Digital interventions and teleaudiology could be a key part of providing high quality, cost-effective, patient-centred management. There is currently very limited evidence that assesses the hearing impaired patient perspective on the acceptance and usability of this type of technology.This study aims to identify patient perceptions of the use of a hearing support system including a mobile smartphone app when used with Bluetooth-connected hearing aids across the everyday life of users, as part of the EVOTION project.We applied a questionnaire to 564 participants in three countries across Europe and analysed the following topics: connectivity, hearing aid controls, instructional videos, audiological tests and auditory training.Older users were just as satisfied as younger users when operating this type of technology. Technical problems such as Bluetooth connectivity need to be minimised as this issue is highly critical for user satisfaction, engagement and uptake. A system that promotes user-controllability of hearing aids that is more accessible and easier to use is highly valued. Participants are happy to utilise monitoring tests and auditory training on a mobile phone out of the clinic but in order to have value the test battery needs to be relevant and tailored to each user, easy to understand and use. Such functions can elicit a negative as well as positive experience for each user.Older and younger adults can utilise an eHealth mobile app to complement their rehabilitation and health care. If the technology works well, is tailored to the individual and in-depth personalised guidance and support is provided, it could assist maximisation of hearing aid uptake, promotion of self-management and improving outcomes. Within the hearing health services, finding ways to support a high quality modern healthcare framework using innovations afforded by the internet and smartphones is central to dealing with ever-rising demands –6.The global increase in prevalence of hearing loss is a key factor in this evolution. Hearing loss is increasingly identified as a public health concern. It is estimated that the number of people with disabling hearing loss (defined as hearing loss >40dB in the better hearing ear for adults and >30dB in the better hearing ear for children) worldwide stands at 466 million and by 2050 is predicted to rise to 900 million . AlthougThis increase in the prevalence of hearing loss will place a further social and economic burden on the hearing impaired population particularly and society in general –10. GlobThe most common intervention to tackle the impact of hearing loss for an individual is a hearing aid and the benefits are strongly evidence based . Howeverpersonalised on-demand service, predictive service through analytics, preventative service through monitoring, and participatory service to empower and reduce costs , received ethical approval from the Research Ethics Committee  and additional local institutional approvals accordingly.There were several different types of tests and activities on the Evotion system. Some of these were designed primarily as data collection tools for the EVOTION study and others were more clearly tools that participants could benefit from directly. The self-administered tests within the EVOTION app aimed to assess factors important for monitoring hearing and listening including cognitive abilities. They included aided pure-tone 4 kHz audiometry, speech-in-babble and digit recall as well as an auditory training module . Speech In the EVOTION system the user had the ability to change the settings on both hearing aids simultaneously . The settings consisted of four listening programs that could be selected by pressing the corresponding button. The programs could be used based on participant preference within different listening environments. The user could also change the volume of the hearing aids up and down using a slider button.The user could access videos showing how to perform hearing aid maintenance tasks, for example changing the minifit dome or receiver wax guard.This test measured pure tone threshold at 4 kHz. Temporary threshold shifts at 4 kHz are indicative of exposure to potentially hazardous loud sounds. The app used a form of sweep audiometry, where the intensity of the tone was varied continuously. If the user could hear the tone, they press the on-screen button and then release when they can no longer hear it. The logged sound environment data then enabled the mobile to calculate the actual level of the tone at the EVOTION HAs and thus calibrate the threshold. The participants were asked to complete this when they felt they were exposed to potentially hazardous loud sounds. Users received instant feedback on the results of the test. The trigger for this was a detected sound level high enough to cause a temporary or permnanet hearing change according to published data .The self-measured 4 kHz PTA was an aided threshold: a probe signal (the tone) was emitted from the EVOTION mobile phone's loudspeaker and then travelled through the air to the EVOTION Hearing Aid, where the level was measured, and the signal was amplified according the patient's audiogram. The EVOTION Mobile phone controlled the intensity (SPL) of the probe tone and recorded patient response as to when the tone was audible and when it was not.The patient was instructed to press the on-screen button when s/he could hear the tone and release the button again when s/he could not hear the tone anymore. They were instructed to place the mobile phone on the table in front of them and to wear both hearing aids during the test. The patient was instructed to keep positions of the phone, the patient and hearing aids constant during the test. During the test the app changed the hearing aid program to the one in which the least amount of processing was applied. At the end of the test the original settings the patient had been using were restored.The mobile app recorded the patient's response and sent the results to the EVOTION server. When the test ended, the mobile app restored the volume settings to those before the audiogram measurements.The test followed the Békésy measurement paradigm where the probe signal was a 4 kHz tone with time-varying level controlled by the EVOTION mobile phone. When Detection thresholds further away from Threshold 1 than This test assessed working memory and cognitive ability. Participants were asked to listen to a sequence of numbers and then type it into the keypad, starting with a two-digit figure and gradually increasing the number of digits. The participants were asked to do the Forward test at least twice and the Backward test twice, once in the first week and once four weeks later.The digit recall test that was implemented in EVOTION was based on the digit span subtest of the Wechsler Adult Intelligence Scale (WAIS) IV . Digits This test was developed based on an existing Speech-in-Babble test , 48 thatThe words in noise test is largely based on the Speech-in-Babble (SiB) test that employs 8 lists of monosyllabic phonemically balanced meaningful English words as the speech stimulus presented with multi-talker babble as the masker. Each list contains 20- 25 words. These are spoken by a female native Southern-English speaker. Each word is delivered with 500 milliseconds of babble masker at the beginning and the end of the word itself .Participants were instructed to place the mobile phone on a table in front of the patient and to wear both hearing aids during the test. The listener was asked to type in the word they heard. The Signal to Noise ratio (i.e. level of the target words vs. noise) is fixed throughout the test at 10 dB and performance was measured with the % correct responses. The English words that were used were those of the SiB test. During the test the app changed the hearing aid progam to the one in which the least amount of processing was applied. At the end of the test the original settings the patient had been using were restored.Equivalent material was recorded in Greek specifically for the purposes of the project based on published Greek phonemically balanced word lists . Words iThis was developed based on the Storey in Noise, an existing auditory training program using words in phrases spoken by adult female and male talkers . PhrasesThe auditory training program in EVOTION was based on the Storey in Noise, that uses words in phrases from connected narratives spoken by adult talkers and presented in background noise .ης, Π. τα), For Whom the Bell Tolls (E. Hemingway) and Perfume (Patrick Süskind). Each text was divided into phrases of 2–10 words. The number of phrases per text ranged from 300 to 2641. The median phrase length for each text was five words. For each phrase, between one and four potential target words were selected. Target words were primarily content words, although function words were used in a small proportion of phrases. Similar sounding foil words were chosen for each target that shared at least two phonemes with the target and were chosen so as to be plausible in the context of the narrative. Phrases were presented with a background of multi-talker babble noise. The participant listened to consecutively presented phrases and after each phrase saw a display containing keywords along with a number of alternatives/foils. Each phrase had up to 4 possible target words. The training ran for a 30 min session, subdivided into four blocks of 7.5 mins. The noise level adapted according to the errors made over the preceding 10 phrases. The initial SNR was set to 10 dB (i.e. target sound 10 dB higher than the noise). If the proportion of possible errors made was > 0.15 then the SNR for the next 10 phrases was increased by 3 dB, otherwise it was reduced by 3 dB.Two texts were implemented in English: “Money for Sale” by M Hardcastle, and “Snowball”, with texts taken from books aimed at foreign learners of English. Three texts were implemented in Greek: Crazy Antonis  participants completed their follow-up appointments as part of the EVOTION protocol. Of these, 564 (54%) filled in and returned feedback forms see . The foup = 0.17). Fisher's exact test indicated no significant difference between clinics in terms of gender distribution (p = 0.176).One way ANOVA indicated no significant difference between clinics in terms of participant age , where there was a significant difference with the oldest and youngest groups reporting higher rates of satisfaction than intermediate age groups  and auditory training (item 7) components, where satisfaction rates were similar see . The UK Feedback questionnaires were given at the follow-up appointment, and participants were under no obligation to provide comments. These were grouped under topic headings that corresponded to the questionnaire items in the quantitative study . Others switched the mobile phone off and on again, and others re-paired the hearing aids with the mobile phone through the Bluetooth setting.Participants in general liked controlling their hearing-aid volume and changing programs remotely, in some cases because the buttons on the hearing aids themselves require better dexterity but also because many people prefer not to draw attention to their hearing aids by touching them in public.A minority of the comments (8% of those about the controls) indicated that people would rather control the hearing aids on the aids themselves.Some people were not aware that they had the option of using the hearing aids to adjust the volumes/programs. For unilateral fittings the volume button was disabled because it could only go in one direction, whereas the app could be used to adjust the volume either way.This was the section of the questionnaire with the fewest and briefest answers. The calibration procedure was reported by some participants to be tricky because of the need for a quiet environment. Most comments discussed limited or non-use of this particular test or the encountering of technical issues completing the test.There were only 35 comments on the instructional videos for the hearing aids included with the mobile phone app. Half were positive, with the rest either non-specific negative comments or comments indicating they had not been used. Nobody criticised the content of the instructional videos. Participants were given clear verbal instructions and issued with printed instruction booklets at their hearing-aid fittings, so the need for instructional videos may have been minimal for some. The “how to change the wax guard” video was the most commonly used, reflecting a common need for an additional hearing-aid appointment.A few people failed to see the relevance of digit recall, which put them off using it. People also found it was very difficult and memorising the digits in reverse order in particular was challenging for many. As with every aspect of the app, some of the comments reported on the activity freezing due to connectivity issues.The purpose of the Speech-in-babble test, an adaptive test that presents a word in background noise, was more apparent to most participants, with some of them commenting on how it related to their real-life difficulties with speech discrimination in noisy environments such as cafes and social groups.A few participants felt that the scoring system was an inaccurate reflection of their ability because they believed they were having to guess. One suggested a progress bar would be a useful indicator to keep the participant motivated.Some participants found the auditory training enjoyable but clear critical themes also emerged including the app crashing; the storey being too dull or long; some participants found it difficult and they felt they were guessing. Connectivity again had a big impact, particularly if the test crashed close to the end of the half-hour timeslot required to do the test, because the participant would have to start again which was disheartening.Many participants questioned the validity of the test because their answers were guesses. However, this test is designed to be a contextual test, reflecting real-life listening environments in which the listener, particularly if hearing-impaired, has to fill in the sound they cannot hear by using contextual and acoustic cues they have heard.Five themes were common to all the auditory activities and are summarised in When asked about their overall satisfaction with using the app with the hearing aids, the overarching message was that people liked having an app that they could use for controlling their hearing aids and they were well-disposed to doing the tests. The connectivity issues became an obstacle to this, however, and in some cases this inconvenience outweighed the benefit of having the device because it made it rather pointless carrying it around, since in the study the app was only available on the phone provided by the researchers and not on the participants' personal devices.Participants wanted to do as well as possible and got frustrated when the tests did not work smoothly. They struggled more with the Auditory Training task than the Digit Recall and Speech in Babble tasks, not least because they were expected to spend far more time on it, in an environment with minimal background noise.Other issues included frustration with the short battery life, which is a result of the phone connection draining the hearing-aid batteries; the frustration of carrying around two mobile phones; and problems for people with poorer eyesight with reading the possible answers on the auditory training tests.Although the questionnaire did not specifically ask about the hearing aids, some of the most positive feedback was based on success with the hearing-aid technology.Participants were positively satisfied across all items , with males and females being equally satisfied, and no difference in satisfaction across the different participating countries.While there were no significant differences comparing age and scoring for most topics there was a statistically significant difference for item 1 (connectivity). This highlighted that the older participants were more satisfied with the connection between the smartphone and the hearing aids than most of the younger participants, except for the very youngest group. The youngest group were a small group relative to the others in the study because hearing loss which mainly affects older adults.This finding around satisfaction with connectivity could indicate that the older group is more adept at dealing with technical issues. However, while previous work has suggested that older aged people are open and eager to adopt technology, they also lack confidence in their ability to use it. They are in general less aware of differences and similarities between different types of technology . It is aThere were fairly consistent differences in satisfaction level between Greek and UK participants with almost all elements, the Greek participants reporting higher levels of satisfaction than the UK participants other than for the audiometry function which the UK participants reported more positively. The reasons for this are not clear, but may have to do with different healthcare systems, different expectations of healthcare or other cultural factors. In any case this illustrates that acceptability of eHealth interventions is not uniform across different ethnic populations. Interestingly, the auditory training task was not rated differently between Greek and English participants, even though the test had components that could potentially be affected by native language.Furthermore, the most common theme was about disappointment and frustration around connectivity. One user said in a very good summary of the overall picture, “I would strongly support the use of this, but for the fact that the connection keeps on failing”. This fits in with previous research that found ease of use and usefulness is important to users and is clearly associated with adoption and intention of use of technology , 56.Controls were highly rated, with 76% of participants reporting satisfied or very satisfied, and there were a large number of positive comments, despite the negative influence of connectivity issues that impacted function. This observation is in keeping with previous studies . ParticiGenerally, users were satisfied with the self-administered hearing and cognitive tests, but conversely if they were not noticing improvement or it lacked usefulness there was a negative effect. This can lead to disengagement with the technology, in keeping with previous research . ImportaLikewise, it was apparent that instruction and feedback of the testing protocol and results needed to be clear and simple in order for the user to understand them and derive meaning. A previous study showed that patients not only experience negative emotions with abnormal results but even with normal results. Simply supplying access to information is counter-productive and guidance is needed on the interpretation . The varThe speech-in-babble test is closer to real world listening situations than the pure-tone-audiogram . Whilst Users did not utilise the 4 kHz audiometry monitoring function very often. The main reason for this according to the patient comments was because it did not work as well as intended. The initial calibration often failed due to insufficient room conditions e.g., high ambient sound level or it crashed. It was not compulsory to complete this test routinely, only if the patient was exposed to an uncomfortably loud sound. A number of users commented here that they “didn't use” or “had not had a reason” to use this test. In relation to this there were very few comments relating to noise exposure even among younger users despite the fact that the app alerts the user when the hearing aids detect loud noise via a notification, and they are advised to perform an audiometry test. The questionnaire did not directly ask about this so it is limited how much we can infer from this. It could be an indication over a lack of awareness within the population about the level of noise that can have a detrimental impact on hearing health. The findings of current research available about awareness of excessive noise levels and noise-induced hearing loss (NIHL) is mixed –68. All A limitation of the study is that it was conducted, by necessity in accordance with ethical practice, on a group of participants who had agreed to use the technology and were aware of this when they signed up to the study. This could mean that the results were obtained on a particularly well-motivated and suited group of participants. However, to combat this limitation the large numbers of participants sampled involved goes some way to obviate this effect and therefore improve the generalisability of the results, since we expect that a range of participants would be included from within this limitation.This study was part of a much bigger project designed to improve hearing care at the public health level and so the suite of tests and interventions had purposes other than direct patient care, meaning that the feedback data described in this study was collected as a cross sectional study without a control group and the EVOTION intervention was not intended as the optimum for patient care. However the results as presented are of interest both in relation to clinical care, public health and also the design of any similar future studies aimed at collecting big data sets for hearing health at the population level.In conclusion, this study showed that 68% of participants were satisfied or very satisfied with overall use of the EVOTION system. Participants rated highly the experience of controlling hearing aids using a smartphone app. Ease of use is a critical factor in satisfaction and design emphasis must be placed on minimising technical issues such as poor connectivity between devices. Negative consequences of poor functionality must also be borne in mind. The majority of users, including the older adults who are the typical demographic within a hearing loss clinic, can utilise eHealth and are willing to adopt a mobile app paired with a wearable to complement their rehabilitation and health care. The older participants reported significantly greater satisfaction with connectivity and there were no significant differences between age, or gender, for all other items of the app including overall satisfaction of use. Although overall satisfaction was high across all groups, there were distinct differences in levels of satisfaction between different participating countries, illustrating that acceptability of technological interventions may vary in different populations. However, if the technology works well, is tailored to the individual and in-depth personalised guidance and support is provided, interventions such as the EVOTION system could be a key tool in maximising hearing aid uptake, promotion of self-management and improving outcomes for the growing health issue of hearing loss.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation on reasonable request.The studies involving human participants were reviewed and approved by London Southeast Research Ethics Committee, NHS Health Research Authority. The patients/participants provided their written informed consent to participate in this study.LM, MS, HW, D-EB, AB, DKi, AO, and NP collected data and contributed to review of the manuscript. HW and MS designed the feedback data collection, collated and analysed the data, and wrote the first draft of the manuscript. LM collated inputs, analysed data, and revised the manuscript. IK and DKo designed and created the app. All authors contributed to the article and approved the submitted version.This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 727521.www.h2020evotion.eu).We would like to thank the participants who contributed to the study, and all the EVOTION partner organisations (list available at The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This retrospective study aimed to investigate the range of hearing levels in a cochlear implant (CI) elderly population receiving electric-acoustic-stimulation (EAS) or electric-stimulation (ES) alone. The investigation evaluates the degree of hearing preservation (HP) and the speech comprehension resulting from EAS or ES-only to identify audiometric factors that predict adequate EAS and ES use. We analyzed the pure tone audiometry and speech perception in quiet and noise preoperatively and 12-months after activation of 89 elderly adults (age of 65 years old or older), yielding in total 97 CIs. Thirty-two (33.1%) patients were potential EAS candidates preoperatively, of which 18 patients used EAS at the time of first fitting and the other 14 patients continued to use their residual hearing for EAS at 12-months. Post-treatment, patients with EAS system and ES-only users’ with longer electrodes showed better results in monosyllable word scores in quiet than ES-only users with shorter electrodes. A similar trend was revealed for the speech recognition in noise. Patients with an EAS system benefit from maintaining their natural residual hearing. Nevertheless, strict preoperative patient selection is warranted particularly in elderly patients, in whom the hearing thresholds for EAS indication differ slightly from that in younger adults. Sensorineural hearing loss (SNHL) is one of the most prevalent chronic diseases among older adults. About one-third of the population in the United States between 65 and 75 years is affected by some degree of SNHL, and the prevalence is estimated to affect more than 50% in the age group 75 years and older ,2,3. SimFor patients with a severe-to-profound SNHL, independent of age, cochlear implants (CI) are the most effective treatment . ImproveDue to increased surgical experience and less traumatic electrodes, the chances of hearing preservation (HP) have improved remarkably. Surgical techniques have been developed to increase HP’s chances and reduce cochlear damage intraoperatively . These tMany studies underlined the influence of the electrode length on HP, showing that the risk of significant postoperative hearing loss (HL) is related to electrode length ,16. In pTo date, studies have compared the results of different age groups altogether and the impact and postoperative performance of EAS, has not yet been studied placing a special emphasis in the elderly population. Our retrospective study sought to establish a treatment plan for elderly population with a high-frequency severe-to-profound hearing loss but good low-frequency thresholds . Another aim of this study was to determine their minimum amount of residual hearing required to achieve a speech-perception benefit from EAS.by (a) assessing the degree of hearing preservation with various thin flexible lateral-wall electrodes; (b) analyzing the speech comprehension of ES-only and EAS users 12-months after surgery; and (c) comparing the pre-and postoperative residual hearing and speech comprehension of ES-only and EAS users. The latter would then provide information about the indication criteria for EAS in elderly patients.This retrospective study was carried out in an academic tertiary hospital for CI and otosurgery based in Germany. Data of 89 patients with 97 CI, aged 65 years and older at implantation, who received a CI in our institution from May 2009 until July 2017, were collected. All subjects had postlingual uni- or bilateral severe-to-profound SNHL in the high frequencies and some residual hearing in the low frequencies. To determine the amount of residual hearing that would lead to the best benefit with EAS in elderly patients, subjects with a preoperative air-conduction threshold better or equal 80 dB at 250 Hz on the implanted side were investigated in the study.The patients were implanted with thin, flexible lateral-wall electrode arrays (TFEA) of different lengths depending on the amount of their preoperative low-frequency residual hearing and cochlear length. The audiometric inclusion criteria for implantation and electrode choice have previously been described ,14.®, Innsbruck, Austria) was performed. Before developing the concept of partial insertion in our clinic, shorter electrodes were selected. Afterwards partial insertion was the preferred treatment option as it allows for deeper insertion of the electrode to provide a higher cochlear coverage for ES only, if hearing is lost after surgery or over time.If patients had significant residual hearing in the low frequencies, meaning 65 dB HL or better at 500 Hz, the patients were informed about the possibility to use EAS by using their preserved residual hearing postoperatively in combination with the CI. If the patients decided on the option of an EAS use, the surgeon decided for a shorter electrode insertion depth intending to preserve the patient’s residual hearing. For an shorter insertion depth either shorter electrode was chosen  or a partial insertion of a longer electrode (TFEA 24 and TFEA 28) , TFEA 20 (n: 18), TFEA 24 (n: 21) and TFEA 28 (n: 51). The partially inserted devices were: TFEA 24 (n: 2), TFEA 28 (n: 2).The CI Surgery was performed using the standardized surgical technique at our department . BrieflyEach patient’s audiometric air conduction thresholds were registered by pure-tone audiometry (measuring the pure-tone average (PTA) low at 125, 250, 500, 1000, and 1500 Hz) preoperatively to define a baseline for each subject. Pure tone audiometer limits were 95 dB at 125 Hz, 100 dB at 250 Hz and 110 dB at 500 Hz to 1500 Hz. Postoperative air conduction thresholds were measured at FF and the 12-months follow-up visit. Preoperative to postoperative change in PTA low for each individual was reported. For analyzing the HP, the postoperative PTA for each visit of the individual subject was plotted against the preoperative PTA. The degree of loss of residual hearing after surgery was divided into three groups: PTA shifts ≤ 15 dB, PTA shift > 15 to ≤30 dB, and PTA shift > 30 dB as described previously .n = 4); subjects with a TFEA 16 electrode (n = 3); subjects with a TFEA 20 electrode (n = 19); subjects with a TFEA 24 electrode (n = 19) and subjects with a TFEA 28 electrode (n = 45).For HP analysis, the subjects were postoperatively divided depending on the electrode array and electrode insertion they received: partially inserted electrodes (TFEA 24 and TFEA 28)  of the CI, approximately four weeks after implantation.n = 6 cases, the crossover frequency was slightly changed to higher frequencies based on patients’ feedback or to better match with the place frequency of the apical contact. In n = 6 patients the postoperative residual hearing was below 65 dB HL in all frequencies, so no crossover frequency was recommended by the software, and the crossover was selected based on the individual slope of the audiogram at a hearing loss between 70 to 80 dB HL.All patients used an individual earmold with tubing for the acoustic amplification, manufactured by a hearing aid acoustician. The acoustic part of the EAS-System and the crossover-frequency between electric and acoustic stimulation were fitted based on the air-conduction thresholds measured at the FF appointment. Initially, the crossover-frequency between electric and acoustic hearing was set at the audiometric frequency at which the HL exceeds 65 dB HL as recommended by the software. In ®, Innsbruck, Austria) in 2017 70 Hz to 8.5 kHz.The subjects using ES-only received a map for ES with a default frequency mapping. The applied frequency range was 100 Hz to 8.5 kHz or since the introduction of the software version MAESTRO 7.0  in the ear to be implanted.n = 6 patientsThe evaluation of postoperative speech recognition was assessed using monosyllabic word test (Freiburg Monosyllabic Word Test (FMWT)) in quiet at 65 dB sound pressure level (SPL) as well as the speech recognition in the German language Hochmair-Desoyer, Schulz, Moser Sentence Test (HSM) in quiet at 65 dB SPL and in noise at 0° azimuth (S0N0) at a 10 dB signal-to-noise ratio (SNR) and post-activation after 12-months of device use. The percentage of correct answers obtained was measured. The tests were conducted with the implanted ear only in the subjects’ everyday listening configuration (EAS or ES). If the patients had a CI or HA on the contralateral side, it was turned off and taken away. Additionally, the contralateral ear was plugged and muffled, in bimodal patients or bilateral EAS patients to eliminate the influence of the residual hearing or masked for a contralateral hearing loss better than 30 dB HL. If not possible, the test was performed using the direct input of the processor, which was the case for Subjects using EAS were tested in EAS-mode; ES-only users were tested with implanted ear open.n = 11), which were treated with shorter electrodes of different lengths  or partially inserted longer electrodes ; the second group consisted of ES-only users with a TFEA 20 electrode (n = 10); the third group consisted of ES-only users with a TFEA 24 electrode (n = 17), and the fourth group consisted of ES-only users with a TFEA 28 electrode (n = 40).For speech test analysis, the subjects were postoperatively divided into four groups whether they used the EAS system or ES only and depending on the electrode array they used for ES only users. The first group consisted of EAS users . All data were analyzed statistically using SPSS .Statistical significance was set to The responsible ethics committee approved the protocol for using the patient’s data for this retrospective study (Project identification code 7657-2018). Due to the retrospective design, no written information was given to the patients of the study group. All patient data were anonymized and de-identified prior to the retrospective analysis.A total of 89 patients, 47 female and 42 male, aged 65 years and older, were implanted at our center. The mean age at implantation was 73 years (65–89-year-old). The patient’s demographic data  are summarized in Thirty-two patients fulfilled the audiometric EAS indication preoperatively. Eight-teen elderly patients tried the EAS-System, 14 subjects were EAS users until 12-months postoperativeTwo patients of those 4 patients, who were no EAS users anymore at 12 months, had suffered from a pre-to postoperative hearing loss > 30 dB and had only minimal residual hearing already at time of first fitting. The EAS system was fitted to try if the acoustic amplification would give at least some additional subjective benefit, which was not the case and they stopped using EAS. One patient with a TFEA 24 electrode used the EAS program additionally to ES, but preferred ES-only. One patient received and EAS-system and used it regularly but was lost to follow-up after the 3 months appointment.n = 3) from 19 dB HL at FF, to 23 dB HL at 12-months (n = 3), for TFEA 24, from 21.3 dB HL at FF (n = 19), to 32.5 dB HL at 12-months (n = 13) and for TFEA 28, from 31.7 dB HL median HL across all frequencies at FF (n = 45), to 34 dB HL at 12-months (n = 15). Hearing was preserved over time for the subjects implanted with a TFEA 20 with a decrease in median HL from 21.3 dB HL at FF (n = 19) to 17.3 dB at 12-months (n = 12)  .FMWT Scores: The median for EAS users was: 45.00%. For the ES-only groups, the median was: TFEA 20 ES: 32.50%, TFEA 24 ES: 50.00%, and TFEA 28 ES: 55.00%.p = 0.015. Post-hoc tests showed a significant difference only between TFEA 20 ES users and TFEA 28 ES users (Bonferroni corrected post-hoc test: p = 0.014). The pairwise comparison of the other groups did not show significant difference  = 3.741, fference A.2.p = 0.033). Bonferroni corrected post-hoc tests revealed a significant difference only between TFEA 20 ES users and TFEA 28 ES users (p = 0.004). The pairwise comparison of the other groups did not show significant difference  = 3.634, fference B.3.p = 0.048. However, Bonferroni corrected post-hoc tests did not show significant difference for pairwise comparisons  = 2.785, parisons C.p = 0.045,), TFEA 24 ES group  and TFEA 28 ES group   .p < 0.001).No difference was identified for age and duration of hearing aid use between the groups. For the preoperative and postoperative hearing thresholds between ES and EAS, significant differences were found along with all analyzed frequencies  and the PTA . All patients using EAS at 12 months had a least some speech perception with headphones preoperatively.The preoperative maximal speech perception with headphones at dBopt, which was in median 100 dB for both groups, was significantly different between the ES (Median: 17.5%) and EAS groups (Median: 32.5%)  CI recipients the degree of postoperative HP rates with outcome comparison of these EAS and ES-only users. The main strengths of this study lie in the large number of cases included, the use of the same atraumatic arrays of different lengths, and the homogeneity of the series of patients: operated by the same team of experienced CI surgeons and underwent the same clinical assessment up to 12-months post activation. We chose this follow-up interval due to the finding of Lenarz et al.  that oneRegarding HP at the FF, the median HL for the partial insertion was 10 dB, for the TFEA 16 19 dB, for the TFEA 20 21.25 dB, for the TFEA 24 21.25 dB, and for the TFEA 28 31.7 dB. In the group of patients implanted with TFEA 16, TFEA 24, and TFEA 28, the median HL increased to 12-months post activation. In the group of patients implanted using partial insertion and TFEA 20, the median HL decreased 12-months post activation. However, a direct comparison of the median HL over time is difficult across all electrodes because the number of subjects undergoing the audiometric assessment at the follow-up dates differed greatly .Moreover, the presented cohort is rather uniform in the criteria for HP, type of electrode arrays, and implemented standardized follow-up protocols; this uniformity further facilitated reliable data comparison with other CI recipients falling in different age groups, as reported in previous studies from our group. Suhling et al.  measuredOur study demonstrates that HP in most elderly CI recipients is feasible 12-months post-activation with shorter electrode arrays—despite concerns about cochlear fragility in these patients. The proportion of the subjects with good HP (≤15 dB) was greater with the shorter insertion  . The losES-only subjects implanted with the shorter arrays, i.e., TFEA 20 and TFEA 24, had higher degrees of residual hearing before surgery compared to the TFEA 28 recipients. We observed that 12 months after CI activation, the HSM in noise test scores of subjects with the TFEA 20 ES-only or TFEA 24 ES-only users who lost usable residual hearing were barely half of those of the TFEA 28 ES group . InteresOur observation of worse HP in elderly patients corroborates the recently published study by Bourn et al. . They anAn explanation for better speech comprehension in the subjects with deeper insertion angles is attributed to a wider cochlear coverage leading to electrically stimulation of the low frequency—apical region in the cochlea ,13,14. AAs the outcomes clearly show, postoperative speech comprehension improved significantly in all elderly CI recipients compared to their preoperative speech perception using HAs. There are additional advantages for EAS users regarding music perception, separating individual talkers from each other, and listening to the intended target speaker ,31,32. TAs reflected in the FMWT scores postoperatively  EAS mighThe above-mentioned factors can be considered preoperatively to select the best treatment solution for elderly patients, as we have provided better understanding on the expected postoperative development of residual hearing and hearing performance, especially with EAS.The preservation of low frequency residual hearing is feasible in elderly CI recipients. The EAS-System for elderly patients is beneficial for natural acoustic hearing, but it needs strict patient selection preoperatively. The preoperative hearing thresholds for sufficient long term EAS use differ up to 20 dB in the frequencies 125 Hz, 250 Hz, and 500 Hz from the younger adults that need to be accounted for. If the residual hearing in the low frequencies is at the limit of the EAS criteria, then ES-only stimulation with longer electrodes leading to deeper insertion enhances the postoperative speech understanding should be preferred in this special patient population.


In the clinical setting, the pathophysiology of sensorineural hearing loss is poorly defined and there are currently no diagnostic tests available to differentiate between subtypes. This often leaves patients with generalized treatment options such as steroids, hearing aids, or cochlear implantation. The gold standard for localizing disease is direct biopsy or imaging of the affected tissue; however, the inaccessibility and fragility of the cochlea make these techniques difficult. Thus, the establishment of an indirect biopsy, a sampling of inner fluids, is needed to advance inner ear diagnostics and allow for the development of novel therapeutics for inner ear disease. A promising source is perilymph, an inner ear liquid that bathes multiple structures critical to sound transduction. Intraoperative perilymph sampling via the round window membrane of the cochlea has been successfully used to profile the proteome, metabolome, and transcriptome of the inner ear and is a potential source of biomarker discovery. Despite its potential to provide insight into inner ear pathologies, human perilymph sampling continues to be controversial and is currently performed only in conjunction with a planned procedure where the inner ear is opened. Here, we review the safety of procedures in which the inner ear is opened, highlight studies where perilymph analysis has advanced our knowledge of inner ear diseases, and finally propose that perilymph sampling could be done as a stand-alone procedure, thereby advancing our ability to accurately classify sensorineural hearing loss. Permanent hearing loss affects more than 5% of the world’s population and is the most common sensory deficit in developed countries . It can The location and fragility of the cochlea poses a significant diagnostic challenge. The cochlea is buried deep within the temporal bone and is surrounded by a thick bony otic capsule, making it difficult to access for diagnostic profiling . ViolatiPerilymph is found in the scalae tympani, scala vestibuli, and in the balance portions of the inner ear and is similar in composition to cerebral spinal fluid (CSF). It bathes multiple structures necessary for signal transduction including spiral ganglion cell bodies, the auditory nerve, the hair cells, as well as portions of the lateral wall of the cochlea and is critical to sound transmission within the cochlea ,11. PeriMany perilymph profiling studies in humans have been performed using postmortem samples. These studies largely form the backbone of our current understanding of the chemical and protein makeup of human perilymph. In fact, postmortem studies were the first to accurately describe ionic concentrations of human perilymph ,15 and iPostmortem perilymph sampling has also allowed for comparisons between CSF, serum, and perilymph, furthering our understanding of inner ear anatomy and the role of each fluid in hearing. Arrer et al. used proteome analysis to compare the levels of α1-antitrypsin and pre-albumin in the CSF, perilymph, and serum. Each fluid demonstrated a different pattern, suggesting that they are distinct fluids with different roles in the inner ear . Later, Although many advances have been made using postmortem sampling, molecular analysis in these studies is limited because of rapid autolysis and degradation of DNA, RNA, and proteins . To overIntraoperative perilymph sampling can also be used to assess direct and systemic drug delivery to the inner ear. Molecules perfused into a perilymph compartment  have direct access to the cells of the inner ear; therefore, optimal drug delivery to the inner ear depends on high concentrations in perilymph. Most studies have focused on quantifying steroid delivery specifically, given that intratympanic and intravenous steroids are commonly used as first line for certain subtypes of SNHL. Using intraoperative sampling during CI, it was found that inner ear levels are dependent on the systemic intravenous steroid dose received , and thaNoise-induced hearing loss (NIHL) accounts for one-third of all acquired cases of SNHL. Exposure to noises greater than 90 decibels for prolonged periods can cause mitochondrial pathology, excessive excitatory neurotransmitter release, and reduced blood flow to the cochlea, all of which result in the production of reactive oxygen species (ROS). ROS then inflict oxidative damage to the cochlear structures necessary for hearing . Perilymph sampling in animal models has been used to measure changes in ROS, cytokines, and other metabolites in the cochlea before and after noise exposure. In mice, one hour of exposure to 110 dB causes a fourfold increase in hydroxy radical concentration in perilymph . In guinAge-related hearing loss (ARHL), also called presbycusis, is the most common subtype of SNHL and has a multifactorial etiology. It was hypothesized by Harman in 1956 that environmental exposures such as noise, ototoxic substances, and age-related cochlear hypoperfusion increase oxidative stress and contribute to the development of ARHL in those with genetic predisposition ,28. ThouIntraoperative perilymph sampling allows for direct comparison of human and animal tissues. It can therefore be used to identify feasible drug targets and guide animal model development. Although there is limited literature comparing animal and human perilymph, there are some studies that compare protein profiles. In 2011, Lysaght et al. found that perilymph from patients undergoing CI for SNHL had 31 orthologs in common with the mouse perilymph profile collected by Swan et al. in 2009 ,30,31. CProtein analysis of perilymph has elucidated the pathways involved in SNHL and has allowed for comparisons between subtypes of SNHL. Mass spectrometry has been used to create and compare comprehensive libraries of perilymph proteins in CI and vestibular schwannoma (VS) patients . AlthougProteins specific to Meniere’s disease (SNHL) have also been identified in human perilymph using liquid chromatography with tandem mass spectrometry. In 2019, Lin et al. compared the perilymph of Meniere’s disease patients to the perilymph of normal hearing patients with skull-base meningiomas and discovered 38 proteins with differential abundance , four ofSome patients with progressive or sudden SNHL are responsive to intratympanic injection of anti-inflammatory medications such as steroids. Therefore, identifying inflammatory protein mediators in the inner ear is of interest to researchers . WarneckStudies of the mouse cochlea in vivo have demonstrated that neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) are essential to neuron survival, and application of these factors can rescue and protect spiral ganglia neurons, which are critical for signal transduction and hearing ,39,40. CHeat shock proteins (HSPs) are thought to protect tissues by refolding denatured or misfolded proteins. They have been implicated in many disease processes, and recently in the pathogenesis of sudden SNHL, Meniere’s disease, and idiopathic SNHL. Serum values of HSPs are significantly higher in those with sudden SNHL compared to normal hearing controls . AdditioCharacterizing the metabolic composition of perilymph fluid is critical for understanding the pathophysiology of deafness and predicting surgical outcomes. This is especially critical for CI patients, as the metabolites in perilymph interact directly with the electrode placed in the cochlea. Perilymph proteome analysis has revealed a relationship between the levels of metabolites such as N-acetylneuraminate, glutaric acid, cystine, 2-methylpropanoate, butanoate, and xanthine and the duration of SNHL . AlthougSimilar to rat, guinea pig, and mouse models, humans with profound SNHL from multiple etiologies display characteristic metabolites in perilymph. In one human study, the perilymph of patients with profound SNHL was compared to that of those with otosclerosis (CHL). Those with profound SNHL had significantly higher superoxide in perilymph and were positive for the ROS-producing enzyme xanthine .MiRNAs are small non-coding RNAs that can be found in body fluids. They serve as reliable biomarkers and prognostic indicators for multiple other neurodegenerative diseases, including Alzheimer’s, amyotrophic lateral sclerosis, and Parkinson’s ,49,50. TIn summary, we can already derive a wealth of information from a sample of perilymph, including predictors of hearing loss, markers of a pro-inflammatory state, factors predicted to protect the inner ear during surgery, and potential predictors of cochlear implant outcomes. Identifying the mechanisms of hearing loss in real time will also allow optimized pharmacologic intervention. To take advantage of this wealth of data, this information needs to be available prior to initiating a planned treatment such as CI. Opening the inner ear was historically considered impossible due to the risk of hearing loss; however, recent experience with hearing preservation cochlear implantation and past sampling studies on stapedectomy patients indicates that it is possible to manipulate the inner ear with no or minimal loss of residual hearing in most patients . We propA cochlear implant is a prosthetic device inserted into the inner ear of patients with severe SNHL and poor speech perception who have minimal improvement with the use of hearing aids. Criteria for undergoing implantation have been recently expanded from including only patients with profound hearing loss to those patients with significant residual hearing but poor speech understanding. The surgery is performed by drilling a mastoidectomy and then entering the middle ear space through a posterior tympanotomy  . The bonCI can improve the speech perception ability in 82.0% of adults with post lingual hearing loss and 53.4% of adults with prelingual hearing loss and can markedly improve quality of life in the responders . HoweverMost of the current perilymph sampling studies have focused on perilymph sampled from cochlear implant patients and information derived from these studies may yield information on optimal pharmacologic intervention to protect hearing during the implantation process. Sampling could also help predict who would be a candidate for supplementation with neurotrophins or who could benefit from drug eluting cochlear implant electrodes . Since pSeveral other operations access the inner ear fluid spaces. Stapedectomy is commonly performed for patients with otosclerosis, a cause of CHL. Stapedectomy is well tolerated and significantly improves hearing, with some studies showing up to 70% of patients achieving an air–bone gap of 20 dB or better ,63. Although opening the stapes footplate accesses the perilymphatic spaces of the inner ear, this approach would probably not be applicable in routine perilymph sampling in patients without a fixed stapes footplate. Additionally, in patients undergoing workup for SNHL, the stapes supra-structure would impede access to the inner ear. Access to the middle ear, which contains the stapes bone, is gained by making an incision in the auditory canal and lifting the tympanic membrane. The bony scutum is then shaved down, allowing for the visualization of multiple middle and inner ear structures, including the round window niche. Stapedectomy is generally not indicated for SNHL and is therefore not useful for directly profiling patients with SNHL. However, it is commonly performed for CHL, which provides an opportunity for a control group. Although sampling during stapedectomy is a debated topic among some clinicians, multiple groups have reported safety using this methodology, and stapedectomy has yielded valuable information on pathogenesis of CHL ,73,74,75The RWM itself is accessed during another open ear surgery called cochleosacculotomy, indicated for patients with refractory MD (SNHL) who have minimal or no residual hearing in the affected ear . In thisFor sampling perilymph, a standard transcanal approach would be used. After making a cut in the ear canal skin, the tympanic membrane is elevated, revealing the structures of the middle ear A. The roSuccessful perilymph sampling depends on developing a device that safely accesses the scala tympani and atraumatically withdraws a small sample. In humans, sterile glass capillary tubes are commonly used for intraoperative sampling via the RWM with preservation of residual hearing ,30,35,52Microneedles can also be used for sampling. There are multiple types, most of which have been developed and optimized in animal models. To collect perilymph, the needle is advanced into the RWM and perilymph is drawn up using a syringe. Multiple studies have shown that perforation of the RWM with microneedles does not affect hearing threshold in animals and are generally atraumatic ,80,81,82Although sampling has been conducted for many years across multiple institutions, there are very few studies directly examining the effects of intraoperative perilymph sampling on post-operative outcomes. Schmitt et al. is the only group to specifically address potential effects on post-CI residual hearing. They compared pre- and post-operative hearing thresholds between patients who underwent CI plus perilymph sampling and randomly selected patients that underwent only CI. No significant differences in residual hearing or speech perception were found between the groups . There is a long history of sampling perilymph in stapedectomy patients. This technique has been used previously for profiling perilymph with no apparent complications but, as noted above, is probably not applicable to routine perilymph sampling for sensorineural hearing loss. Some additional insight can be gained from the surgical procedures in which the inner ear is opened. Stapedectomy is considered a safe procedure having only a minimal incidence of SNHL . HoweverThere are also some technical limitations of the sampling procedure. Studies in guinea pigs show that perforation of the RWM induces perilymph outflow driven by CSF pressure, leading to possible CSF contamination of the sample, and that samples greater than 10 μL can be significantly contaminated with CSF . There cNew diagnostic techniques are needed to further subclassify SNHL. As proposed here, a round window membrane “tap” can be performed to profile perilymph and determine SNHL subtype and candidacy for adjunctive medical treatment with cochlear implantation. Although perilymph sampling has been performed intraoperatively in humans for decades, it remains controversial due to paucity of literature on post-operative effects on hearing, and further safety studies are needed.