Legs with asymptomatic varicose veins exhibited lower MF for the GSV (p = 0.02) compared with legs with symptomatic varicose veins. Among legs with varicose veins, asymptomatic legs exhibited lower ASV (p = 0.03) and MF (p = 0.046) for the GSV compared with legs that exhibited skin changes or ulcers; however, no significant differences were observed between legs presenting with discomfort or edema and legs with skin changes or ulcers, and between legs presenting with discomfort or edema and asymptomatic legs. In conclusion, in the supine position, increased blood flow rate and blood flow volume in the GSV were associated with symptomatic varicose veins and increased symptom severity.The progression of clinical manifestations of lower-limb varicose veins remains unclear. This study investigated changes in lower-limb venous blood flow using phase-contrast magnetic resonance angiography. Data were collected on veins from 141 legs. We compared legs with and without varicose veins and related symptoms and examined varying levels of varicose vein symptom severity. Legs without varicose veins exhibited a lower absolute stroke volume (ASV,  Lower-limb varicose veins are typically related to chronic venous insufficiency and pathologic changes to the venous walls ,2 and caAlthough the gold standard for evaluating varicose vein severity is color-flow venous duplex ultrasound, the advent of three-dimensional phase-contrast magnetic resonance angiography (PC-MRA) has increased the ability to inspect venous anatomy from the feet to the pelvis, in addition to providing an easier and more intuitive method for measuring the venous blood flow rate and volume. However, the patient is required to maintain a supine position during MRA scans, which can influence the accuracy of estimations for venous reflux by removing the ability to evaluate the influence of gravitational forces on lower-limb venous reflux. Therefore, in this study, we aimed to investigate whether any associations exist between changes in lower-limb venous blood flow and the severity of clinical manifestations of varicose veins in the supine position.The protocol for this retrospective study was approved by the Institutional Review Board of Chang Gung Memorial Hospital (CGMF IRB No. 202100680B0). The requirement for obtaining written informed consent was waived for this study due to the retrospective and anonymous nature of the patient data used.In this retrospective study, we collected data on 214 legs from all 107 patients who underwent PC-MRA as part of a lower-extremity vascular survey conducted between October 2018 and December 2020. Legs without complete blood flow data and legs that did not contain varicose veins but exhibited symptoms similar to venous disease were excluded. Ultimately, the data from 141 legs were used in this study.Baseline demographics and clinical characteristics included age, sex, comorbidities, and clinical inspection. The abnormal venous anatomies found in the PC-MRA were collected, and included compression of the iliocaval territory, deep vein thrombosis, dilatation of the great saphenous vein , in addition to the GSV to popliteal vein (PV) diameter ratio. The PV diameter was used to normalize the GSV diameter to decrease inherent differences in venous size among different people because deep vein dilatation is uncommon. The GSV and PV diameters were measured at the same height using the frontal view of three-dimensional, maximum-intensity projection images. Venous blood flow was analyzed using the quantitative flow (QFlow) technique, which collected the blood flow parameters, including the regurgitation fraction (RF), absolute stroke volume , mean flux (MF), and mean velocity (MV) of the external iliac veins (EIVs), femoral veins (FVs), PVs, and GSVs.In addition to comparing the baseline demographic and clinical characteristics between patients with and without varicose veins, this study used two steps to analyze the lower-limb venous anatomy and blood flow data. The first step was to classify the legs into the following groups: (1) legs without varicose veins; (2) legs with asymptomatic varicose veins; and (3) legs with symptomatic varicose veins. The second step was to classify the legs with varicose vein by their clinical presentations into three groups: (1) no symptoms; (2) discomfort  or edema (C3 using the CEAP classification system); and (3) skin changes (C4 using the CEAP classification system) or ulcers (C5 and C6 using the CEAP classification system).Among currently available non-contrast magnetic resonance imaging techniques, our hospital adopts triggered angiography non–contrast-enhanced magnetic resonance imaging . A sequence of three-dimensional turbo spin-echo techniques and a short tau inversion recovery protocol were used to separate venous structures from background tissues and arterial signals. Blood flow measurements were performed with the QFlow application . DetailsContinuous variables are presented as the median and interquartile range, whereas categorical variables are presented as the number and percentage. To compare two independent groups, the Mann–Whitney U test was used to analyze continuous variables, and the Chi-square test or Fisher’s exact test was used to analyze categorical variables. To compare three or more independent groups, the Kruskal–Wallis test, followed by Dunn’s test for post hoc analyses, was used to analyze continuous variables, and the Chi-square test and pairwise Z test  were used to analyze categorical variables.All statistical analyses were conducted using SPSS for Windows .The median age of enrolled patients was 62 years (interquartile range: 53–74 years), with women forming the majority (66.4%). Patients with and without varicose veins are compared in p < 0.001). In addition, the post hoc analyses showed that legs with symptomatic varicose veins had higher values for the GSV to PV diameter ratio (p = 0.001), the GSV ASV (p = 0.004), and the GSV MF (p = 0.026) than legs without varicose veins. Legs with symptomatic varicose veins had a higher GSV MF value than legs with asymptomatic varicose veins (p = 0.02). However, legs without varicose veins and legs with asymptomatic varicose veins had similar GSV to PV diameter ratios and similar GSV blood flow parameters. The line plot also displays a substantial increase in the median GSV ASV and GSV MF values in legs with symptomatic varicose veins relative to both legs without varicose veins and legs with asymptomatic varicose veins , no significant differences were observed between legs with discomfort or edema and legs with skin changes or ulcers . Furthermore, post hoc analyses indicated that legs with skin changes or ulcers had higher values for the GSV to PV diameter ratio, GSV ASV, and GSV MF than legs with asymptomatic varicose veins; however, no significant differences were observed between legs with discomfort or edema and legs with no symptoms, and between legs with discomfort or edema and legs with skin changes or ulcers. Therefore, the values of the GSV to PV diameter ratio, GSV ASV, and GSV MF may increase with increased varicose vein progression . The line plot also showed that the GSV ASV and GSV MF of legs with discomfort or edema appear in a transition state between legs with no symptoms and legs with skin changes or ulcers . Changes in varicose vein blood flow may have little to no influence on the blood flow in deep veins, suggesting that the loss of ability to modulate the balance of blood flow between the superficial and deep veins may represent another important factor. The connection between the superficial and deep veins is known to involve the perforator veins. Past studies have demonstrated that incompetent perforator veins are associated with the severity of chronic venous disease, and superficial venous reflux can deteriorate perforator vein competence ,20. HoweIn the QFlow data analysis, the GSV RF did not increase with the increasingly severe clinical manifestations, which was unsurprising because magnetic resonance imaging must be performed while the patient is in a supine position, removing the influence of gravitational force and muscle contractions on lower-limb venous blood flow ,25. HoweMany other factors are likely to influence symptom severity, such as the baseline characteristics of sex, age, and body mass index . Patientthe lack of consideration of behavioral factors, family history, and doppler ultrasound evaluation in the assessment of venous hemodynamic changes is one of the limitations of this study.This study primarily used PC-MRA data collected from patients in the supine position. Generally, the severity of venous reflux is determined by the reflux duration observed in the standing position using venous duplex ultrasound. Therefore, PC-MRA results are likely to underestimate RF values. In addition, In the supine position, increases in GSV blood flow rate and volume are associated with the development of symptomatic varicose veins and, potentially, the progression of symptom severity. Our findings may help physicians prevent and predict the progression of varicosity-related symptoms.This project is under the reviewing process in the Taiwan Intellectual property Office. (No 109126307).


The growing aging population with disabilities poses challenges to caregiving and health care services but there is little recent data on disability trends. Some studies have shown that disability is decreasing while others have shown it increasing. Understanding these trends among community-dwelling older adults is critical for communities to allocate resources and develop policies. This study updates disability trend data among community-dwelling older adults using nationally representative National Health & Aging Trends Study data. Results revealed that about 30% of Medicare beneficiaries had at least one limitation of the activity of daily living (ADL) from 2011 to 2019. Age 75-79 (IRR=1.55), 80-84 (IRR= to 4.60), 85-89 (IRR=2.99), 90+ (IRR=4.60), female, (IRR=1.18), not a house owner (IRR=1.51), financial strain (IRR=1.71), and receiving Medicaid (IRR=1.84) are associated with a higher likelihood of becoming ADL limited or having more ADL limitations. We will discuss potential policy, intervention, and research implications.


Glucosamine, an intermetabolite of the hexosamine biosynthesis pathway (HBP), is a widely used nutritional supplement in osteoarthritis patients, a subset of whom also suffer from diabetes. HBP is activated in diabetic retinopathy (DR). The aim of this study is to investigate the yet unclear effects of glucosamine on DR.in vivo and on cultured endothelial and Müller cells to elucidate the underlying mechanisms of action in vitro.In this study, we tested the effect of glucosamine on vascular and neuronal pathology in a mouse model of streptozotocin-induced DR 1c levels in the animals, but induced body weight gain in the non-diabetic animals. Interestingly, the impaired neuronal function in diabetic animals could be prevented by glucosamine treatment. Correspondingly, the activation of Müller cells was prevented in the retina as well as in cell culture. Conversely, glucosamine administration in the normal retina damaged the retinal vasculature by increasing pericyte loss and acellular capillary formation, likely by interfering with endothelial survival signals as seen in vitro in cultured endothelial cells. Nevertheless, under diabetic conditions, no further increase in the detrimental effects were observed.Glucosamine did not alter the blood glucose or HbAIn conclusion, the effects of glucosamine supplementation in the retina appear to be a double-edged sword: neuronal protection in the diabetic retina and vascular damage in the normal retina. Thus, glucosamine supplementation in osteoarthritis patients with or without diabetes should be taken with care. •The hexosamine biosynthesis pathway (HBP) is activated in diabetic retinopathy (DR), which manifests as vascular and neuronal damage in the retina.•Glucosamine, metabolized in the HBP, is a widely used oral supplement for osteoarthritis treatment.•Glucosamine supplementation improved neuronal function in retinas of mice with experimental DR, but induced vascular damage in normal retinas.•Müller cell activation and endothelial survival signals in the retina were affected by glucosamine.•Patients with or without diabetes should take caution with glucosamine supplementation. ACAcellular capillariesAng2Angiopoietin 2DRDiabetic retinopathyECEndothelial cellERGElectroretinogramGFAPGlial fibrillary acidic proteinGOTGlutamic oxaloacetase transaminaseGPTGlutamic pyruvic transaminaseHBPHexosamine biosynthesis pathwayHRMVECsHuman retinal microvascular endothelial cellsHUVECsHuman umbilical vein endothelial cellsO-GlcNAcO-linked N-AcetylglucosamineOCTOptical coherence tomographyPCPericyterMCsRat Müller cell line;UDP-GlcNAcUridine diphosphate N-Acetyl glucosamine;VEGFVascular endothelial growth factorVEGFR2Vascular endothelial growth factor receptor 21The hexosamine biosynthesis pathway (HBP), a nutrient-sensing pathway, accounts for 2–5% of the total glucose flux under normal conditions  and combDiabetic retinopathy is a long-term microvascular complication of diabetes. In its early stage, diabetic retinopathy is characterized morphologically by the loss of pericytes and subsequent disappearance of endothelial cells from capillaries. This leads to the formation of acellular capillaries that consist of only the vascular basement membrane and do not support blood flow, resulting in the progression of the disease . The patBiochemically, glucosamine is an intermetabolite of the HBP, an offshoot of glycolysis in glucose metabolism. It stimulates glycosaminoglycan production and the incorporation of sulfur into cartilage . For thiin vitro.Diabetes and osteoarthritis often occur concomitantly in elderly patients. Additionally, exogenous glucosamine supply, though it ameliorates cartilage damage, might increase protein O-GlcNAcylation in the retina and thus aggravate diabetic retinopathy. Therefore, we sought to determine the effects of oral glucosamine supplementation in an experimental diabetic retinopathy model in mice and further elucidate the mechanisms of action by studying its effects on endothelial and Müller cells 22.1The use of mice in this study was approved by the local ethics committee . The care and experimental use of the animals were in accordance with institutional guidelines, in compliance with the Association for Research in Vision and Ophthalmology (ARVO) statement, and reported in compliance with the ARRIVE guidelines. The mice were housed in a 12-hour dark/light cycle with unrestricted access to food and water and under the supervision of trained staff. Diabetes induction in the mice was performed via intraperitoneal injection of streptozotocin (145 mg/kg body weight) into 8-week-old male C57Bl/6 mice, and glucosamine treatment was commenced after 1 week for up to 24 weeks. Glucosamine was provided to the animals via incorporation into food at a concentration of 10 g/kg . Metabol2.2-1. Baseline separation of amino acid derivates was achieved by increasing the concentration of acetonitrile (B) in buffer A as follows: 1 min 8% B, 7 min 9% B, 7.3 min 15% B, 12.2 min 18% B, 13.1 min 41% B, 15.1 min 80% B, hold for 2.2 min, and return to 8% B for 1.7 min. Data acquisition and processing were performed with the Empower3 software suite (Waters).Non–thiol-containing amino acids were quantified after specific labeling with the fluorescence dye AccQ-TagTM (Waters) according to the manufacturer's protocol. The resulting derivatives were separated by reversed phase chromatography on an Acquity BEH C18 column  connected to an Acquity H-class UPLC system and quantified by fluorescence detection . The column was heated to 42 °C and equilibrated with 5 column volumes of buffer A  at the flow rate of 0.45 mL min2.3Retinal digestion was performed to isolate retinal vessels, and PAS staining was done to quantify the retinal cells as previously described  using th2.4d-Glucose in ECBM/ECBM MV or DMEM for HUVECs/HRMVECs and Müller cells, respectively. Glucosamine stimulation in the cells was performed by preparing a stock solution of 100 mM glucosamine  in ECBM/DMEM immediately prior to stimulation and diluting according to the required concentration in the experiments.The use of Human Umbilical Vein Endothelial Cells (HUVECs) was approved by the local ethics committee . HUVECs were isolated from umbilical cords obtained from healthy newborns with the informed consent of their mothers using a previously described protocol . HUVECs 2.52 and 1 cd/m2 for the m-sequence with four dark frames in between the stimuli. An average of eight cycles was used for each hexagon for the final analyses, which were processed by the built-in 50-Hz band filter to reduce background noise. For each animal, the average amplitude of the 6 hexagons around the optic nerve head was used for final analyses. mfERG recording took place under photopic conditions wherein both rod and cone photoreceptors were activated in mice. The initial negative-going N1-wave was initiated by photoreceptors, whereas the following positive-going P1-wave was generated in the inner retina, mainly by ON-bipolar cells under the influence of Müller cells. Optical coherence tomography (OCT) was subsequently performed using the built-in OCT of the RETImap system. Retinal thickness was measured at five locations along the border between the inner third and the outer two-thirds of the retina.Multifocal electroretinography (mfERG) was performed as previously described . The mic2.6The eyes were fixed in 4% paraformaldehyde on ice for 2 h. The retinas were dissected and washed in PBS for 3 h and incubated in permeabilization/blocking solution (3% w/v BSA and 0.1% Triton-X in PBS) for 1.5 h at room temperature. The retinas were incubated with primary antibody  overnight at 4 °C. Post-incubation, the retinas were washed with PBS for 3 h and incubated with the secondary antibody solution  for 1.5 h at room temperature. The retinas were subsequently washed with PBS, cut into 4 leaves, mounted on glass slides using Roti FluorCare mounting medium, and covered with a coverslip. The retinas were imaged using a Leica SP8 confocal microscope. Four fields per retina were randomly chosen and z-stack images spanning 80 μm were obtained. The images were visualized using a 3D viewer, displaying the GFAP staining in astrocytes on the superficial layer and in Müller cells spanning the depth of the retina. Astrocytes were quantified visually in the superficial layer, and the lengths of the GFAP staining in individual Müller cells were quantified per field and compared between groups.2.7HUVECs and rMCs were cultured according to experimental conditions and subsequently washed with PBS and fixed with 4% Histofix for 10 min, after which they were incubated with a permeabilization/blocking solution (2.5% BSA and 0.3% Triton-X in PBS) for 1 h at room temperature. The cells were further incubated with 200 μL of primary antibody solution  overnight at 4 °C, then washed thrice with PBS and incubated with the secondary antibodies  for 1 h at room temperature in the dark. The cells were subsequently washed thrice with PBS, incubated with DAPI (1 μg/mL) for 10 min, and washed twice with PBS. They then were covered with Roti FluorCare mounting medium and covered with coverslips. The images were obtained using a Leica SP8 confocal microscope, and fluorescence intensity quantification was done using ImageJ.2.8Proteins from HUVECs and rMCs were extracted in RIPA buffer using a previously described method . The pri2.9The data are presented as mean ± SD. Statistical analyses were performed with GraphPad Prism 6. Paired/unpaired student's t-test or one-way ANOVA with Tukey's multiple comparison test were used. p values < 0.05 were considered statistically significant.33.11c levels reflect the average level of blood sugar over the past 120 or 40 days in humans and mice, respectively. Correspondingly,  was used to measure the thickness of the mouse retinas. The retinal thickness remains unaltered between the groups, showing no changes with glucosamine supplementation A. FurtheThe electrical activity of the retina in response to a light stimulus, as assessed by electroretinography (ERG), gives an indication of the neuronal function of the retina. The different waveforms generated in the ERG correspond to the activity of different cell types in the retina. The N1-wave of the ERG, generated by photoreceptors, showed no significant changes between any of the groups under photopic conditions B. In the3.3Müller cells are glial cells in the retina that perform several functions, including modulation of the neuronal microenvironment and facilitation of crosstalk between the vasculature and the neuronal compartment. We first examined the expression of GFAP in the retina. Under confocal microscopy, we reconstructed the three-dimensional architecture of GFAP-positive cells in the retina. In the superficial layer, astrocytes were stained positive for GFAP. In the non-diabetic retinas, GFAP expression can be seen mainly in astrocytes in the ganglion cell layer, which display normal morphology and are distributed evenly but sparsely throughout the retina A. In thein vitro , we investigated the expression of GFAP with high glucose and glucosamine stimulation in vitro A,B. Stroin vitro D,E.FigurIn addition, we examined the expression of VEGF in the rMCs. Müller cells are a prominent secretor of VEGF in the retina, contributing to vascular regulation. HG did not alter VEGF expression compared to the normal glucose control. Glucosamine treatment was, however, able to significantly reduce the VEGF protein level in the rMCs A,C. The 3.4Pericyte dropout and the formation of acellular capillaries are the hallmarks of vascular damage in the retina, and the quantification of their coverage in the retinal vasculature provides an indication of the initiation of retinal vascular injury. To assess the vascular damage in the retinas, the numbers of pericytes and acellular capillaries were quantified in retinal digest preparations stained with PAS A. Compar3.5in vivo with glucosamine supplementation, we assessed the impact of glucosamine on cultured endothelial cells. VEGF-VEGFR2 signaling is crucial to endothelial cell survival. Additionally, Ang2, a key growth factor that mediates pericyte loss [In order to obtain insights into the vascular damage observed yte loss , is prodElevated production of Ang2 from endothelial cells is a cause for the pericyte loss in the retinal vasculature . Thus, wWe substantiated our findings with immunofluorescence staining of VEGFR2 and Ang2 in HUVECs under normal and high-glucose conditions G. As witIn addition to HUVECs, we confirmed our findings in endothelial cells of retinal origin—human retinal microvascular endothelial cells (HRMVECs). The HRMVECs were treated with increasing doses of glucosamine, with or without high glucose. As in HUVECs, the HRMVECs showed a decrease in VEGFR2 A,B,D,E a4In our study, we demonstrate that glucosamine treatment in diabetic mice can improve the function of Müller glial cells, inducing a neuroprotective effect. However, it causes vascular damage in the retina, inducing pericyte loss and acellular capillary formation in non-diabetic retinas, which might be a result of interfering with endothelial survival signals. Our findings suggest that oral supplementation with glucosamine can elicit multiple countenances in the retina, including previously unknown risks.in vivo.To the extent of our knowledge, this is the first study demonstrating the effects of dietary glucosamine supplementation on neuronal and vascular function in diabetic retinopathy in vitro [In accordance with previous studies ,32, our in vitro . The disin vivo glucosamine treatment. Examining GFAP expression in a rat Müller cell line in vitro, we observed that glucosamine treatment reduces GFAP expression and hence Müller cell activation. Contrary to previous studies [in vitro was in a state of constant activation that could not be further stimulated by high glucose; glucosamine was nevertheless able to reduce this activation, resembling the reduced GFAP expression in vivo. Activation of Müller cells can have both beneficial and detrimental effects in the diabetic retina. Müller cells are the source of major survival signals for neurons in diabetes [Based on our data, which show an improved P1-wave amplitude following glucosamine treatment, we further focused on Müller cells in the retina. In accordance with published data , we foun studies , we did diabetes . Activatdiabetes . Our finin vitro in endothelial cells (both HUVECs and HRMVECs) despite the elevation of protein expression from high glucose, indicating that glucosamine-provoked vascular damage is presumably not initiated by pericyte loss. Ang2 regulation can be controlled by the cellular O-GlcNAc cycle [In opposition with the protective effects conferred by glucosamine on the neuronal function in the retina, its effects in the vasculature seem to be more detrimental. The damaging effect of glucosamine on the vasculature seen in our study supports studies showing that glucosamine consumption is correlated with the development of arteriosclerosis, CVD risk, and mortality, in which case endothelial dysfunction upon glucosamine supplementation may play a role . In the Ac cycle . HoweverAc cycle , suggestAc cycle . Ang2 caAc cycle . VasculaGlucosamine treatment in our study did not induce changes in blood glucose or HbA1c. This information is in line with several clinical trials in humans that also show that glucosamine lacks an adverse impact on blood glucose and HbA1c levels  or on glWe can therefore show that glucosamine exerts multifaceted effects on the retinal neurovascular unit, both protective and detrimental. It exerts a neuroprotective effect in the diabetic retina, but can induce vascular damage by suppressing the expression of VEGFR2 and Ang2 in normal retinas. As a widely used oral supplement, glucosamine requires caution for proper use. Further research needs to be performed in order to determine whether the benefits of glucosamine outweigh the risks.YF and TW conceived and designed the study. RE, LT, KM, MK, GJ, DZ, and GP performed experiments and acquired data. RE, YF, LT, MK, DZ, HPH, and MS analyzed and interpreted the data. RE and YF wrote the paper with input from the other authors. All authors gave final approval for publication.10.13039/501100001659Deutsche Forschungsgemeinschaft .This work was supported by grants from the


Severe sensorineural hearing loss can be a symptom of the benign tumor vestibular schwannoma (VS). The treatment of VS with non-invasive stereotactic radiosurgery (SRS) offers a high local tumor control rate and an innovative possibility of sequential hearing rehabilitation with cochlear implantation. This study evaluated the feasibility, complications, and auditory outcomes of such a therapeutic approach. Three males and one female (mean age 65.3 ± 9.4 years) scheduled for cochlear implantation and diagnosed with sporadic VS classified as T1 or T2 (according to Samii) were enrolled in this study. All patients had progressive hearing loss qualifying them for cochlear implantation. First, the tumor was treated using CyberKnife SRS. Next, sequential auditory rehabilitation with a cochlear implant (CI) was performed. Clinical outcomes and surgical feasibility were analyzed, and audiological results were evaluated using pure tone audiometry and speech recognition tests. All patients exhibited open-set speech understanding. The mean word recognition score  improved after cochlear implantation in all four patients from 5.0 ± 10% (with hearing aid) preoperatively to 60.0 ± 22.7% six months postoperatively. Our results suggest that in patients with profound hearing loss caused by sporadic vestibular schwannoma, the tumor removal with SRS followed by cochlear implantation is an effective method of auditory rehabilitation. In rare cases, vestibular schwannoma (VS) can induce profound hearing loss or deafness, a condition qualifying the patients for cochlear implantation. VS are benign neoplasms originating from Schwann cells surrounding the 8th cranial nerve as a myelin sheath . The oveFor patients with deafness or profound hearing impairment, cochlear implantation offers a possibility for auditory rehabilitation. A treatment plan should be established before implantation if an incidental finding of VS’s or ILS’s occurs during the cMRI evaluation. According to the accepted guidelines and recently published studies ,9,10,11,SRS for the treatment of VS was introduced in the 1980s by Lecksell . Since tHearing loss is a common symptom of VS’s, but the reasons for hearing loss in VS patients are still not fully understood. The widely accepted view assumes dysfunction of or damage to the eighth cranial nerve or its peripheral branches. This hypothesis is supported by the pathological findings in auditory brainstem response and the cochlear nerve atrophy ,16,17,18This report discusses sporadic VS, concomitant functional deafness, CyberKnife treatment, and sequential CI hearing rehabilitation. The innovative concept of SRS and CI for patients with sporadic VS and hearing loss is presented and evaluated regarding feasibility and auditory outcomes.The local Ethics Committee approved this retrospective study , which was conducted at a tertiary referral center to review VS treatment and hearing rehabilitation methods. All patients signed written informed consent. Three males and one female (mean age 65.3 ± 9.4 years) were retrospectively included in this study. The patients were diagnosed with profound sensorineural hearing loss and referred to our Cochlear Implantation Unit by secondary care specialists. All patients expressed their intention to undergo sequential hearing rehabilitation after SRS. “Sequential” was defined as cochlear implantation performed weeks, months, or years after SRS. All patients were diagnosed with sporadic VS. Neurofibromatosis was excluded on clinical and molecular pathology levels. Clinical diagnostics included collecting focused medical history, an ear, nose and throat (ENT) examination, vestibular examination, and bedside head impulse test (bHIT). In addition, videonystagmography and caloric testing were performed. Audiological performance was tested with pure tone audiometry (PTA), and the word recognition score WRS performed with Freiburg Monosyllabic Test (FMT) with numbers and monosyllables in quiet at 65 dB SPL (sound pressure level) with a hearing aid before surgery or with the CI speech processor after surgery. The objective audiological assessment was performed before surgery using auditory brainstem responses (ABR). In case of negative ABR in the affected ear, an in-house subjective hearing nerve test was performed. During that test, an electrode is placed in the external auditory meatus, and electrical stimulation is used to determine the integrity of the auditory nerve. During the stimulation, rectangular alternating current electrical stimulation is applied for 500 ms. The three frequencies used are 62 Hz, 125 Hz, 250 Hz. The stimulation begins at 100 μA, and the current is increased manually up to 1000 μA. In most patients with preserved residual hearing, stimulation with 200–300 μA induces an acoustic sensation of humming. The amperage causing the hearing sensation is designated as the electrical hearing threshold (EHT). After defining the EHT, the current is increased further until an intense, unpleasant sensation of a sound occurs (designated unpleasant hearing level—UHL). The difference between the EHT and the UHL is called dynamic.Oldenburger Sentence Test (OLSA) was used to assess hearing with background noise after implantation . OLSA coRadiological diagnostics included cranial computed tomography (CT) of the temporal bone, and magnetic resonance imaging (cMRI) acquired with a 1-mm slice thickness using intravenous gadolinium contrast. The MRI protocol included T1- and T2-weighted sequences, T1 VIBE (volumetric interpolated breath-hold examination), fat-saturated post gadolinium sequences, and T2w SPACE sequences and/or 3D CISS (constructive interference in steady-state) sequence. The cMRI-imaging identified the VS in all cases. According to the Samii  grading Patients # 1–3 were incidentally diagnosed with VS during pre-implantation diagnostics. All patients experienced progressive hearing loss, resulting in profound sensorineural hearing loss or deafness in the VS-affected ear. Patients # 1, 3, and 4 were bilaterally deaf, whereas Patient #2 had asymmetric hearing loss see . All patAfter discussing the clinical findings and treatment options during the interdisciplinary Skull Base Board meeting and with the patients, three patients  opted for primary CyberKnife radiosurgery. Their reasons for deciding against the microsurgical removal were advanced age, comorbidities and/or small tumor size. Patient # 2 decided for secondary Cyberknife treatment for residual VS after initial surgical treatment. Unilateral implantation was performed in all patients after CyberKnife radiosurgery. The following cochlear implants were used: (CI) model CI 512 (patient #1), MedEl Synchrony Flex 28 (patients # 2 and 4), and Advanced Bionics Hires Ultra 90K (patient # 3). The implantation procedure was successful in all patients; no symptoms of cochlear fibrosis or post-radiation changes in the middle and inner ear after SRS were observed. n = 4). Six months postoperatively, the mean aided WRS  with CI was 60.0 ± 22.7% . One year postoperatively, the mean aided WRS  with CI was 46.7 ± 12.6% . The mean preoperative aided WRS with a hearing aid at 65 dB (FMT) was 5.0 ± 10%  or CyberKnife treatment, cMRI to verify tumor control, and sequential CI surgery. The patient chose the second option and underwent CyberKnife SRS  for the right ear affected by the tumor. Six weeks after SRS, cochlear implantation (CI 512) was performed on the contralateral left ear. Two years later, after cMRI demonstrating the unchanged status of VS, the patient was scheduled for CI surgery (CI 512) on the right side. Hearing rehabilitation was successful, and six months after implantation, the patient achieved an aided WRS of 55% (FMT at 65 dB) and a number recognition score  of 90% on the right side. One year after the second cochlear implantation, the WRS was 75% (FMT at 65 dB) with binaural CI. The number recognition score (FNT at 65 dB) with binaural CI was 100% (6 months and one year postoperatively).Hearing in noise was measured with the Oldenburg Sentence test (OLSA) with noise and speech presented from the front. Six months postoperatively, this test revealed scores of −0.3 dB signal-to-noise ratio (SNR) for the right side and −3.7 dB with bilateral CI, indicating an improvement of hearing in noise with CI.A 61-year-old male patient was referred for hearing rehabilitation with CI on the left side because of progressive asymmetric hearing loss see  and limiContrast-enhanced cMRI revealed an intra- and extracanalicular VS (Samii T2) A,B. PossOne month after implantation, aided WRS with CI on the left side (and masking of the right side) was 45% (FMT at 65 dB SPL) and six months later, 60%. Binaural hearing with CI on the left side and hearing aid on the right side resulted in an aided WRS of 90% (FMT) with CI only after two years.Hearing in noise was measured with the aided Oldenburg Sentence test (OLSA). One year postoperatively, this test revealed scores of −1.1 dB signal-to-noise ratio (SNR) for the left side with CI (with masking of the right ear) and −3 dB with CI and hearing aid on the right side, indicating an improvement of hearing in noise with CI.A 76-year-old male complained of bilateral progressive hearing loss for approximately 35 years see  and recuCranial MRI performed during evaluation for implantation revealed a multilocular schwannoma on the right side: small IC VS (T1) and a small intracochlear schwannoma A,B. The A 57-year-old female patient presented with IC VS on the right side with profound hearing loss after SRS. At the age of 4, she had mumps resulting in a profound sensorineural hearing loss on the left side. An earlier CI evaluation revealed a negative promontory test on the left side. In 2010, she developed hearing loss on the right side, and IC VS was detected using cMRI. SRS was performed in 2019 in a different hospital (3 × 6 Gy) to stop tumor progression and prevent further hearing loss B, and byHearing in noise was postoperatively measured with the aided Oldenburg Sentence test (OLSA). One year postoperatively, the patient scored 1.5 dB signal-to-noise ratio (SNR) with unilateral CI. This study presents four sporadic VS cases and demonstrates that the innovative concept of SRS and CI for patients with sporadic VS and hearing loss is feasible and safe and that the auditory performance of implanted patients improves significantly. This concept cannot only be used for the therapy of small intracanalicular VS (patient #1), but also patients with surgically removed VS (patient #2), with multilocular VS and intracochlear location of VS (patient #3), or progressive hearing loss after SRS (patient #4). The use of SRS for VS therapy increases ,13. In pAmoodi et al. , CostellIn our present study, auditory rehabilitation with CI was successful in all patients, and the mean WRS  improved from 5.0 ± 10% before implantation to 60.0 ± 22.7% six months postoperatively. Patient #4 scored 90% in the speech recognition test of the monosyllabic words at 65 dB (FMT). Interestingly, hearing rehabilitation with CI seems very effective after SRS with preserved cochlear nerve, and hearing outcomes are comparable to non-irradiated CI ears. The postoperative aided WRS in the discussed studies ranged from 28% to 96%. Therefore, we assume that SRS might not necessarily damage the cochlear nerve fibers, and the auditory nerve is still functional. The degree of damage to the cochlea and the cochlear nerve resulting from SRS and their long-term consequences are still not fully understood. However, one may assume that essential prognostic factors for good postoperative hearing rehabilitation and WRS include a short duration of deafness or residual hearing and preserved cochlear anatomy without fibrosis. The pathomechanisms of hearing loss in patients with VS are still not fully understood. Still, it is essential to take them into account to understand why cochlear implantation should be considered for patients with VS and NF 2. Various theories explain the pathomechanisms of hearing loss accompanying these diseases, and multifactorial etiology is likely of importance there. One explanation of hearing loss could be tumor-compromised perfusion of the inner ear tissues by a labyrinthine artery. In addition, there is evidence suggesting the occurrence of cochlear changes in patients with VS, such as pathological otoacoustic emissions  and histFurthermore, there are theories about cytokines and TNFα causing SHL in the ears with VS . DilwaliDespite the theories of cochlear changes in ears with VS, it is crucial to remember that SRS may also cause damage to the cochlear structures and the cochlear nerve. In particular, high radiation doses seem to be a risk factor. There are reports of a higher risk of hearing loss after SRS with higher radiation doses and larger irradiated cochlear volumes . The cocAfter CyberKnife radiosurgery for VS, local tumor control is estimated to be >95% two years after treatment and >90% after ten years post-treatment ,39. In sn = 1), 12 (n = 2), and 24 (n = 1) months postoperatively, and heterogeneity of cases regarding tumor size and location. The small number of patients is due to the limited time window in which this innovative concept of SRS and cochlear implantation was performed. Moreover, only bilaterally hearing-impaired patients were included. There are few published cases of NF 2, SRS, and CI in the literature and even fewer sporadic VS, SRS, and CI cases. Therefore, this study aims to create awareness for this particular topic and to add to the knowledge of the earlier published cases. Further knowledge should be generated by extending the sample size and designing a multicenter study.The major limitations of our study include a small number of patients, the limited follow-up time of the auditory outcome of 6 . Additionally, improved communication is a significant advantage, even though it is unknown how long the hearing nerve will remain functional and if the patient can still hear with a CI after many years or decades. Therefore it would be interesting to perform a long-term follow-up study regarding this topic.Cochlear implantation offers an option for successful hearing rehabilitation in selected patients with profound hearing loss associated with vestibular schwannoma or induced by stereotactic radiosurgery of vestibular schwannoma.


Zygomycetes class of fungi, which carries significant morbidity and mortality. While typically associated in patients with underlying immunocompromise , it may also be seen in immunocompetent patients. We report a case of a healthy 4-year-old girl with acute right leg cellulitis and abscess formation, who required surgical debridement following poor response to antibiotic therapy and initial incision and drainage. Tissue histopathology returned cutaneous zygomycosis despite negative tissue cultures. At four-week follow-up, her wound was healed well. Clinicians should maintain a high degree of clinical suspicion for cutaneous mucormycosis given its potential for rapidly progressive and disseminated disease. Currently, the mainstay of diagnostic investigations is tissue histopathology, with a growing role for tissue fungal PCR. Treatment involves multidisciplinary management between surgeons and Infectious Diseases team to guide the role for surgical debridement and antifungal therapy.Cutaneous mucormycosis is a rare infection by the  Zygomycetes class of fungi, normally found in soil and decaying vegetation [Cutaneous mucormycosis is a rare infection caused by the getation . Other tgetation . Cutaneogetation ); thus, 9/L with predominant neutrophilia and lymphocytosis, and C-reactive protein was 23 mg/L. The right leg X-ray was unremarkable, and ultrasound found a 31 × 12 × 28 mm area of subcutaneous oedema, with no drainable collection.A 4-year-old Caucasian girl presented to a regional hospital with a three-day history of the right leg cellulitis, after sustaining a minor splinter from burr grass while playing in a paddock. She was an otherwise healthy child with no relevant medical history. On examination, she was febrile but hemodynamically stable, with a localised 3 × 3 cm area of cellulitis over the distal anterior surface of her right leg. There were erythema and induration, but no obvious fluctuance. On investigation, white cell count was 17.6 × 10She was commenced on intravenous cefotaxime, flucloxacillin, and vancomycin. Despite antibiotics, her cellulitis progressed, and a fluctuant collection developed. On day 2, she underwent an incision and drainage of the abscess where purulent fluid was expressed, and the wound was packed. Postoperatively, she had persistent low-grade fevers and ongoing purulent discharge. Thus, on day 3, she underwent a further debridement and washout of the wound. Intraoperative findings included a sloughy, devitalised tissue mass  which waTissue histopathology returned positive for zygomycosis with necrotic adipose tissue and suppurative inflammation . At one-Apophysomyces, Saksenaea species, and Lichtheimia corymbifera species are relatively frequently reported causative agents in trauma-related mucormycosis [This case highlights some important practice points regarding cutaneous mucormycosis infection. Firstly, cutaneous mucormycosis typically follows direct inoculation, such as after trauma, burns, or surgery. Previous outbreaks of cutaneous mucormycosis have been also associated with natural disasters and combat-related injuries . Apophysrmycosis . Howeverrmycosis , 5. As srmycosis . While trmycosis ), a signrmycosis , 7–10. OSecondly, clinicians should maintain a high degree of clinical suspicion for cutaneous mucormycosis to aid with timely diagnosis. Wound cultures are often falsely negative, and there are no rapid diagnostic serology/PCR tests currently available . MaintaiThirdly, the hallmark of cutaneous mucormycosis infection is tissue necrosis, due to angioinvasion and intravascular thrombosis. Complications include deep tissue spread, necrotizing fasciitis, and disseminated disease. Thus, the mainstay of treatment is aggressive  surgical debridement, as supported by the 2019 consensus guidelines for the diagnosis and management of mucormycosis . RepeateTo conclude, we report a rare case of cutaneous mucormycosis in an immunocompetent patient who had no predisposing risk factors, other than the recent innocuous skin trauma. The cornerstones of management remain prompt aggressive surgical debridement to prevent dissemination to deeper tissues, antifungal agents, and optimisation of underlying immunosuppression.


Laboratory and clinical-based assessments of speech intelligibility must evolve to better predict real-world speech intelligibility. One way of approaching this goal is to develop speech intelligibility tasks that are more representative of everyday speech communication outside the laboratory. Here, we evaluate speech intelligibility using both a standard sentence recall task based on clear, read speech (BKB sentences), and a sentence recall task consisting of spontaneously produced speech excised from conversations which took place in realistic background noises (ECO-SiN sentences). The sentences were embedded at natural speaking levels in six realistic background noises that differed in their overall level, which resulted in a range of fixed signal-to-noise ratios. Ten young, normal hearing participants took part in the study, along with 20 older participants with a range of levels of hearing loss who were tested with and without hearing-aid amplification. We found that scores were driven by hearing loss and the characteristics of the background noise, as expected, but also strongly by the speech materials. Scores obtained with the more realistic sentences were generally lower than those obtained with the standard sentences, which reduced ceiling effects for the majority of environments/listeners (but introduced floor effects in some cases). Because ceiling and floor effects limit the potential for observing changes in performance, benefits of amplification were highly dependent on the speech materials for a given background noise and participant group. Overall, the more realistic speech task offered a better dynamic range for capturing individual performance and hearing-aid benefit across the range of real-world environments we examined. Among the primary functions of speech-in-noise testing are the prediction of speech intelligibility and device benefit outside the clinic or laboratory conditions in which testing is conducted. However, numerous studies have identified discrepancies between the results of speech testing and self-reported speech understanding and device benefit in everyday settings . For exaOverestimation of real-world performance has led researchers to identify the need for more challenging speech tests . HoweverTo create speech tests which can provide more generalizable results it is necessary to account for the cause of overestimation of real-world performance, rather than finding arbitrary ways to make speech tests more challenging. A potential cause can be seen if we consider the differences in perceptual cues provided to listeners by clear speech of the type employed in speech test materials, and conversational speech that is frequently encountered in daily life. Like any complex signal originating in the environment, speech signals consist of multiple redundant cues . These cBy this logic, one approach to improving the predictive capabilities of speech testing is to incorporate features of conversational speech, such as phonetic reductions and realistic speech rates, into the test materials. Including features found in conversational speech has the dual benefit of increasing both the difficulty and realism of speech tests. We recently took this approach in developing the Everyday COnversational Sentences in Noise (ECO-SiN) test . The ECOOur expectation is that the naturalistic aspects inherent to the ECO-SiN sentences will make them less intelligible than clearly articulated sentences typical of existing speech tests. However, at the same time, their vocal effort is appropriate for situations involving background noise, which should enhance the SNR at mid to high frequencies . The potTo better understand the effect of using more realistic speech materials on hearing outcomes, we directly compared the intelligibility of the highly realistic ECO-SiN sentences to that of more traditional sentences when each were presented in six different realistic background noises. The speech and noise signals were presented at their realistic (fixed) levels (and thus SNRs) and performance was quantified by the percentage of words correctly recognized. Our evaluation included young listeners with normal hearing as well as older listeners with hearing loss, who are ultimately the target population for new and more effective approaches to speech testing. Listeners with hearing loss were assessed unaided and aided to also determine the effect of hearing-aid amplification on speech scores. The outcomes of this exploratory study are intended to highlight the advantages (and possible disadvantages) of increasing the realism of the speech materials in the assessment of speech perception in realistic background noise.t-tests found no significant differences in age between the three groups with hearing loss (p > 0.1) but showed that 4FAHLs were significantly different (p < 0.05 using Bonferroni corrections). Ten young adults with normal hearing (NH) and 20 older adults with hearing loss were recruited as part of a larger study. All participants reported that they were native Australian-English speakers and had no known cognitive or neurological problems. The NH group had audiometric thresholds below 20 dB HL at all audiometric frequencies between 250 and 8,000 Hz. The requisites for admission into the group with hearing loss were symmetrical sensorineural hearing loss with no more than one audiometric pure-tone threshold differing by more than 10 dB between the ears. Four frequency  average hearing loss (4FAHL) was calculated for each individual, and participant groups were established based on the following criterion according to The realistic sentence materials were drawn from the ECO-SiN corpus cf. . The ECOThe more traditional (reference) materials were drawn from a corpus of “BKB-like” sentences created by the Cooperative Research Centre for Cochlear implant and Hearing Aid Innovation (CRC HEAR). These sentences are similar to the original BKB sentences , howeverThe average spectrum of the speech materials is shown in The corresponding temporal modulation spectra of the different speech materials are shown in the right panel of The background noises were drawn from the ARTE database , which wThe speech levels for the six different environments see  were derNote that the speech levels given in The loudspeaker signals for the different noise and speech conditions were transformed into binaural signals by simulating their playback via the 41-channel loudspeaker array to the in-ear microphones of a Bruel and Kjaer  type 4128C Head and Torso Simulator (HATS). Additionally, to enable the integration of a pair of hearing aids in the binaural playback, behind-the-ear (BTE) hearing aid satellites were placed above the left and right ear of the HATS. These purpose-built satellites were provided by Sonova AG  and included front and rear microphones that were connected to a purpose-built pre-amplifier. The playback simulation path included individual loudspeaker equalization filters as well as measured impulse responses from each of the 41 loudspeakers to the six microphones at the left and right ears of the HATS: two in-ear microphones and four hearing aid microphones. However, only the front hearing aid microphones were used in this study to realize an omni-directional hearing aid input. Further details of the playback simulation process can be found in HLP, to mimic the passive attenuation of the hearing aid fitting as well as a headphone equalization filter, HEQ. The equalization filter ensured a flat frequency response of the headphones when measured on the HATS. The aided path describes the signal path from the hearing aid microphone via the hearing aid processing to the headphones. This path includes (1) a BTE microphone to free-field transformation filter, B2FH, that removes the acoustic head shadow for a frontal sound source and provides a free-field equivalent output; (2) a multi-channel wide dynamic range compressor (WDRC) as the main hearing aid processing; (3) a free-field to ear-drum transformation filter, F2EH, that basically reintroduces the acoustic head shadow for a frontal sound source but as recorded by the in-ear microphone; (4) a high-pass filter to simulate the limited sensitivity of the hearing aid receiver at low frequencies; (5) the same headphone equalization filter used in the acoustic path; and (6) an instantaneously acting broadband limiter, Lim, to protect the listener from excessively loud sounds.A standard desktop computer was used to run the listening tests and to play the different 4-channel speech and noise stimuli via a RME Fireface UC  USB sound card to a second desktop computer with an RME Audio Fireface UFX USB sound card. The second computer ran a real-time hearing-aid research platform developed at the National Acoustic Laboratories, Hearing Australia, and presented the (aided) binaural stimuli to the participants via Beyerdynamic  DT990 headphones. All stimulus playback was realized at a sampling frequency of 44.1 kHz except for the hearing aid platform, which operated at a sampling frequency of 24 kHz and was band-limited to about 10 kHz.HLP, and high-pass filter, HHP, shown in Lim, was part of the sound card of the hearing aid platform and was set to an attack time of 0 ms, a release time of 100 ms, a compression ratio of 6, and a knee-point of 95 dB SPL. The limiter was significantly engaged only for the participants with moderate and moderate-severe losses, and then only in the loudest environments. For NH participants as well as participants with hearing loss in the unaided conditions, materials were presented through the acoustic path only, with the lowpass filter removed  noise and speech levels, and thus SNRs see . The senThe order of the six background noises and the two speech materials  was randomized. These test conditions were blocked for the participants with hearing loss within the unaided and aided conditions due to the required manual reconfiguration of the hearing-aid platform. The two blocks were tested in random order.To quantify the effect of speech material (BKB vs. ECO-SiN) on unaided and aided speech intelligibility scores, a Bayesian Beta regression model was fitted  using thHearing-aid benefit was calculated by subtracting the unaided speech intelligibility percentage score from the aided speech intelligibility percentage score for each individual, separately for the BKB and ECO-SiN materials, with positive values indicating that amplification provided an improvement in speech intelligibility. Mean benefits are shown in the bottom row of Given the complex behavior of the unaided and aided scores described in section “Speech Intelligibility Scores,” the differences between them were also complex and were strongly affected by floor and ceiling effects. The largest aided benefits were observed for the listeners with moderate-severe hearing loss in the quietest environments. In those same environments, ceiling performance tended to reduce or eliminate the measurable benefit for better-performing listeners with milder losses. For the louder environments , floor effects meant that benefits of amplification were generally not observed for the listeners with moderate-severe hearing losses. In these louder environments though, better performing listeners who were not at floor demonstrated negative benefits (or “disbenefits”). In some cases, the magnitude of the benefit clearly depended on the type of speech material used.disbenefits for the ECO-SiN materials.To quantify the effect of speech material (BKB vs. ECO-SiN) on hearing-aid benefit, a robust regression model with a Student-T noise distribution was fitted to model hearing-aid benefit data which is not constrained to the  interval. The results of this analysis are provided in A quantile regression model was fitted to compare the behavior of the individual ECO-SiN scores as a function of BKB scores in each environment and aiding condition with the predicted slopes describing the relative spread of the distributions of ECO-SiN and BKB scores. Quantile regression was used because it is robust to outliers and makes no assumptions about the underlying distribution of the data. The regression lines in p < 0.001), church , living room , and café  environments. In the two loudest environments there is a trend toward lower rates of change in ECO-SiN scores relative to BKB scores, with a slope of 0.75  in the dinner party environment and a slope of 0.56  in the food court environment. In the aided conditions, a similar trend is seen with slopes becoming progressively shallower in the louder environments. We see high relative rates of change in ECO-SiN scores in the four softest environments including the office , church , living room  and café . Very low relative rates of change in ECO-SiN scores occurred in the two loudest environments including the dinner party  and the food court .In unaided conditions, ECO-SiN and BKB scores show very similar spreads in the office  that change drastically across the different test conditions  in the three softest environments: the office, church, and living room. Clustering around zero on the x-axis was less clear in the café and dinner party environments. In the loudest environment, the food court, there was instead evidence of clustering of data around zero on the ECO-SiN benefit scale (y-axis).Also shown in In this study we demonstrated that by using sentences embedded in a range of real-world environments, with their natural SNRs, the overall difficulty of a speech-in-noise test can be varied in a meaningful way. This means that by selecting the right environment a useful operating point (where scores are away from both ceiling and floor) can be found for listeners across a wide range of hearing abilities. Depending on the specific purpose, the test environment may be selected based on the individual’s hearing loss, their reported speech-in-noise problem, or the relevance of a test environment  has important consequences if the aim is to examine the effect of a particular intervention. In our study, this point was made for the case of non-linear hearing-aid amplification. Because intelligibility scores varied substantially across environments, degree of hearing loss, and speech material, so too did the ability to measure a benefit of amplification. For instance, as shown in negative benefits were observed for the listeners with mild hearing loss in the louder environments. In these conditions, where the overall SNR is negative, speech audibility is not expected to play a significant role because the main limitation is the presence of the noise. Accordingly, it is unsurprising that amplification did not provide any strong improvement in intelligibility. Moreover, the distorting effects of compression, limiting and/or microphone placement may have had a negative impact on intelligibility by reducing the effective SNR at the listener’s ears  adaptive beamforming e.g., . IncludiThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Macquarie University Human Research Ethics Committee and the Australian Hearing Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.KM: design and conceptualization, data curation, analysis, and writing the manuscript. TB: design and conceptualization, analysis, and writing the manuscript. VB: analysis and writing the manuscript. JB: design and conceptualization, analysis, writing the manuscript, and supervision. All authors contributed to the article and approved the submitted version.The authors declare that this study received funding from Sonova AG. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.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.


P < 0.05). Anti-phospho-Ser276 (Ser276-P)-labeled plaques were significantly increased in LIM retinas at both 12 h and 7 days. The density of Ser276-P of Cx35 was observed to increase after 12 h LIM. In the meanwhile, the areas of existing Cx35 plaques did not change. As there was more phosphorylation of connexin35 at Ser276 at both the early and late stages (12 h) and 7 days of LIM chicken retinal activity, the coupling with ACs could be increased in myopia development of the cone-dominated chicken retina.Our previous research showed that increased phosphorylation of connexin (Cx)36 indicated extended  coupling of AII amacrine cells (ACs) in the rod-dominant mouse myopic retina. This research will determine whether phosphorylation at serine 276 of Cx35-containing gap junctions increased in the myopic chicken, whose retina is cone-dominant. Refractive errors and ocular biometric dimensions of 7-days-old chickens were determined following 12 h and 7 days induction of myopia by a −10D lens. The expression pattern and size of Cx35-positive plaques were examined in the early (12 h) and compensated stages (7 days) of lens-induced myopia (LIM). At the same time, phosphorylation at serine 276  of Cx35 in strata 5 (S5) of the inner plexiform layer was investigated. The axial length of the 7 days LIM eyes was significantly longer than that of non-LIM controls ( Myopia or nearsightedness is the most common refractive error and usually results from excess axial elongation. Its prevalence has been increasing rapidly, with over 80% of adolescents in Hong Kong and 40% of individuals older than 12 years in the United States being myopic  provides a reliable and rapid model to study mammalian eye growth and human refractive development. The chick model has been used for over 40 years  and the state of Cx35/36 phosphorylation . The phosphorylation state of Ser276/293 indicates the degree of coupling through Cx35/36 gap junctions in the myopic retina. Cx35/36 phosphorylation leads to an increasing gap junction coupling between AII ACs in the myopic mouse retina  of the Hong Kong Polytechnic University and complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.Gallus gallus domesticus) were hatched from specific pathogen-free eggs (China) (n = 28).White Leghorn chickens  and sampled at 250 Mhz with Agilent 1067A Acqiris DP110 Digitize (Resolution up to 50 µm) respectively. Streak retinoscopy  was used to measure refractive error. Refractive errors and biometric measurements were conducted both before and after minus lens wear. Retinoscopy was used to measure the refractive status.Rabbit anti-phospho-Ser276  was used for retinal labeling  and blocked with 3% donkey serum in 0.1 M PB 0.5% Triton-X 100 and 0.1% NaN3 overnight. The antibodies were diluted in 0.1 M PB with 0.5% Triton-X 100 and 0.1% NaN3, containing 1% donkey serum. The tissues were incubated in primary antibodies for 3–7 days at 4°C and, after extensive washing, incubated in secondary antibodies overnight at 4°C  for observation.For consistency, all chicken retinas were taken from the pecten’s dorsal side (only the middle section) around 10 AM under illuminance (500–700 Lux). The retinal pieces, attached to filter paper (RGCs up) were submersion-fixed in 2% N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride , pH 7.5 for 30 min at room temperature. After fixation, the retinas were separated from the filter paper and washed with phosphate buffered saline (PBS) before being washed extensively with a 0.1 Chicken retinal proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes by standard procedures. After blocking with Tris-buffered saline containing 0.1% Tween-20 and 5% skim milk powder for 1 h at 37°C, the membranes were incubated with primary antibody overnight at 4°C. Mouse anti-glyceraldehyde-3-phosphate dehydrogenase  antibody was used as a loading control.3, 0.8 Na2HPO4, 0.1 NaH2PO4, 1 MgCl2,2 CaCl2, and 5 d-glucose. The bath solution was continuously bubbled with 95% O2–5% CO2 at 32°C. A piece of central retina was embedded in type VII agarose (Sigma) and cut with a vibratome (Leica VT1200S) to 200 μm thickness. Retinal slices were transferred to the recording chamber and superfused with the oxygenated extracellular solution (flow rate 1–2 ml/min) for neurobiotin injection.The retina was carefully separated from the sclera in a chamber with extracellular solution, containing (in mM): 120 NaCl, 2.5 KCl, 25 NaHCO3 containing 1% donkey serum and then, after extensive washing, incubated in Cy3-conjugated streptavidin  1:200 overnight at 4°C. The tissues were mounted in Vectashield (Vector Laboratories) for observation.The AII-like ACs identified were similar in their morphology to those of the mouse retina, having thick lobular appendages in the OFF sublamina of the IPL and descending arboreal processes to the ON sublamina of the IPL  were superimposed. Z-axis steps were usually 0.35 μm. Based on the optical resolution of 120 nm in the x- and y-axes. Four fields from each retina  were analyzed. The retinas were imaged under identical acquisition conditions, including laser intensity, pinhole, photomultiplier amplification, and z-stack step size. The detector gains and offset parameters were optimized. All the settings remained unchanged after optimization for each experiment to avoid imaging biases. The region of interest (ROI) identification was accomplished by using Image J software . ROI borders were defined by a 20% intensity threshold in the channel and a size threshold of 0.01 μm2 as well. The analysis was conducted as described previously  confocal microscope using a 63× objective (N.A. 1.4). The instrument’s optical resolution was 120 nm in the P < 0.05) was determined by using Wilcoxon Signed Rank test.Statistical analyses were performed using Origin software  and SPSS version 25 . Statistical significance , respectively    of the cP = 0.36, n = 7)    and AL  .As phosphorylation of Cx35 at Ser276 is involved in the regulation of coupling in retinal neurons, Ser276-P antibodies were used to visualize phosphorylated Cx35 and, thereby, identify coupled gap junctions. The specificity of Ser276-P antibodies was determined with Western blots against phosphorylated Cx35 proteins in the chicken retina , demonstrating that the antibody was highly specific for Cx35 in the chicken retina –3C. TherAlthough AII ACs are well characterized in the mammalian retina  (n = 5) , the middle and distal INL and the ganglion cell layer in the whole mount-retinas (n = 3) . Prox1-in = 3) . Followi (n = 5)  and 4D, n = 5) (Most Cx35 puncta (98.7 ± 0.04%) were located in the IPL of the chicken retina. Ser-276 (96.7 ± 0.03%) puncta were also predominantly located in the same layer . Cx35 aln = 5)  and 4F. 3 μm2) between LIM myopic and non-LIM control retina (n = 11) and 7884 plaques in non-LIM control eyes (n = 11), a significant difference in the area of S276 plaques was observed .The eye growth had fully compensated for −10D defocus after 7 days, the other eye as control with the Plano lens. Ser276-P antibody recognized the Ser276-phosphorylated form of Cx35 in S5 of the IPL of the chicken retina was observed after 7 days LIM. There was no significant difference in density  and plaques in non-LIM control eyes (n = 11), no difference in the area covered per punctum of Cx35 across groups was observed  and control retina was observed   than controls   . Followi = 0.02) .Fig. 7.Q3 μm2 in the control retina did not differ significantly from the LIM retina, the percentage of Ser-276 of Cx35 phosphorylated was higher in LIM myopic retinas than non-LIM controls. Following evaluation of plaques in retinas of LIM and control eyes, no significant difference in the area covered per Cx35 plaque across groups was observed  implies that gap-junctional coupling is extended. Therefore, the increase in phosphorylation of Ser293 in AII-ACs in the myopic mouse retina indicates that extended coupling of AII ACs involved in the myopia development of mammals. The AII AC is an interneuron cell in the scotopic (night-vision) pathway in the retina and also contributes to photopic (daylight vision) in cone-dominated primate retinas . The amount of phosphorylation of Ser276 and the size of Ser276 phosphorylated Cx35 plaques were significantly increased after 12 h in LIM chicken myopic retinas  compared with control mouse retinas. This may suggest that phosphorylation of Cx35/36 was involved with the coding of differential signaling of myopic/hyperopic defocus. We further hypothesize that myopia development started in the retina where AII-like ACs might act as a midway to deliver the signaling of defocus from RGCs to the sclera.In conclusion, this study showed an increase in phosphorylation of Cx35 in the cone-dominant retina of the LIM myopic chicken. Further studies are needed to clarify the complex etiology and nature of physiological remodeling in myopia development.Chicken does not employ the same retinal circuitry as mammals, which have a specialized rod pathway via the unique rod bipolar AII AC-cone bipolar cell network. AII, A8, and A17 ACs may only exist in mammalian species. Even Prox1-immunoreactive AII-like ACs were identified and indicated the possible AII ACs in the chicken retina. AII-like ACs were still named in the research. Because of the difference of retinal circuit, findings from the chicken cannot be matched with that of the mouse. The gap junction state (Cx36) in the research was estimated from the phosphorylation of Cx36. However, this is an indirect estimation, and future studies involving gap junction permeable tracer coupling and electrophysiological recording need to be done.Therefore, the findings from the chicken myopia model should be cautiously applied to human myopia research.


Mixed metal oxide (MMO) is one of the widely utilized ceramic materials in various industries. In order to obtain high performance, the specific surface area of MMO should be controlled. Calcination of layered double hydroxide (LDH) is a versatile way to prepare MMO with homogeneous metal distribution and well-developed porosity. Although researchers found that the specific surface area of LDH-originated MMO was relatively high, it had not been systematically investigated how the surface area is controlled under a certain parameter. In this review, we summarized LDH-originated MMO with various starting composition, calcination temperature, and pore developing agent in terms of specific surface area and porosity. Briefly, it was represented that MMOs with Mg-Al components generally had higher specific surface area than Mg-Fe or Zn-Al components. Calcination temperature in the range 300–600 °C resulted in the high specific surface area, while upper or lower temperature reduced the values. Pore developing agent did not result in dramatic increase in MMO; however, the pore size distribution became narrower in the presence of pore developing agents. Due to the variation in metal composition, even ternary or quaternary metal composition is possible . I. I59]. Ie itself . Zhu et e itself  prepared2/g) than conventionally prepared LDH (78 m2/g). As expected, the AMOS-MMO also had much higher SBET (325 m2/g) than MMO without acetone treatment (256 m2/g) . The high SBET value in this example is also interpreted as the enhancement in interparticle cavity. The hierarchical layer-stacking structure of AMOS-LDH could be robustly maintained during calcination preserving the particle-particle distance, finally maximizing the adsorption site.The AMOS-LDH itself had higher specific surface area value  ,63,64. Dectively . This inectively , we coulBET values of ZnAl-MMO ranged between 45 m2/g to 85 m2/g, exhibiting relatively narrow distribution compared with MgAl-MMO. Huang et al. ..48].BET (224.3 g/m2) than MMO from coprecipitated albumin-LDH (SBET = 176.6 m2/g). This was attributed to the different degree of aggregation of albumin depending on synthetic condition; reconstruction reaction preserves constant pH value, resulting in the homogeneous distribution of albumin as porogen. Thus, the reconstructed MMO contained well-developed inter-particle pore  and excellent catalytic activity. Self-templating synthesis of MMO could be an alternative method to evolve mesopores and enhance specific surface area of MMO. Chen et al. . transfo2O4 spinel exhibited high specific surface area of 324 m2/g. The organic glucose contributed to the evolution of mesopores and high specific surface area .Glucose template was another approach to generate mesoporous MMO. Li et al.  proposedBET increase at moderate temperature: (i) formation of intraparticle pore coming from the degassing and (ii) interparticle space which is strongly governed by the interaction of particles.Application and utility of MMO is increasing in various fields such as catalysts, electrodes, adsorbents, construction, environment, etc. due to their diverse composition and high specific surface area. One of the recent approaches to prepare MMO is calcining LDH; thermal treatment on LDH gives rise to dehydration, simultaneous dehydroxylation, and degassing of anion, and development of the spinel phase depending on temperature. According to the literature survey, calcination at moderate temperature 300–600 °C resulted in pure MMO with fairly high specific surface area. Calcination at lower or higher temperature resulted in the mixture of  or (spinel + MMO) to decrease the specific surface area. It is expected that there are two reasons of SBET, MgAl-MMO generally tended to show relatively large specific surface than ZnAl-MMO (45–85 m2/g), while MgFe-MMO had wide range of SBET. This may be highly related to the surface energy of divalent metal oxide (MgO or ZnO) and site preference of Al3+ and Fe3+. In order to achieve very high SBET, specific synthetic process such as AMOST, water-n-BuOH solvent, water-in-oil microemulsion method were applied; in this way, thinner and small pristine LDH particles could be prepare and the inter-particle space would be well-preserved to enhance high specific surface area. Calcination temperature was determined to be related with specific surface area, showing generally increasing SBET upon temperature. In addition, the utility of pore-developing agent was also adapted in LDH-originated MMO. Although the SBET value did not dramatically increase in the presence of porogen, it could develop homogeneous porous structure, which is advantageous in adsorbent or catalyst.As for the metal composition effect on S


Likewise, for a four-frequency pure tone average calculated at 500–1,000–2,000–4,000 (0.5–4 kHz PTA), an increase of 4.5 dB HL was measured (p = 0.002), and this increase was significantly greater than that seen for 0.5–2 kHz PTA. There was no difference in the average age of the control and AD subjects. These data confirm the presence of poorer hearing ability in AD subjects, provided a quantitative estimate of the magnitude of hearing loss, and suggest that the magnitude of the effect is greater at higher sound frequencies.An association between age-related hearing loss (ARHL) and Alzheimer's Disease (AD) has been widely reported. However, the nature of this relationship remains poorly understood. Quantification of hearing loss as it relates to AD is imperative for the creation of reliable, hearing-related biomarkers for earlier diagnosis and development of ARHL treatments that may slow the progression of AD. Previous studies that have measured the association between peripheral hearing function and AD have yielded mixed results. Most of these studies have been small and underpowered to reveal an association. Therefore, in the current report, we sought to estimate the degree to which AD patients have impaired hearing by performing a meta-analysis to increase statistical power. We reviewed 248 published studies that quantified peripheral hearing function using pure-tone audiometry for subjects with AD. Six studies, with a combined total of 171 subjects with AD compared to 222 age-matched controls, met inclusion criteria. We found a statistically significant increase in hearing threshold as measured by pure tone audiometry for subjects with AD compared to controls. For a three-frequency pure tone average calculated for air conduction thresholds at 500–1,000–2,000 Hz (0.5–2 kHz PTA), an increase of 2.3 decibel hearing level (dB HL) was found in subjects with AD compared to controls (Systematic Review Registration:https://www.crd.york.ac.uk/prospero/, identifier: CRD42021288280. As the sixth leading cause of death in the United States, Alzheimer's disease (AD) affects nearly 6.2 million Americans  and the subsequent development AD is increasingly well-documented; however, the nature of this relationship remains unclear ” resulted in a total of 417 studies; no languages were excluded. All citations containing information on author, title, source, and full abstract were exported from each database as a BibTeX file and uploaded to Mendeley® citation manager for preliminary review. Once duplicates were removed, the abstracts, methods, and results of 248 studies were individually screened. Off-topic studies were removed first and the remaining studies were assessed using the inclusion/exclusion criteria set by this study. This was completed by one rater (SSK) and repeated by two raters (XMTN and DDW) for validation.A systematic review of literature using PRISMA guidelines was conducted on studies published prior to August 18th, 2021 using PubMed, Cochrane Library, Web of Science, and Scopus databases  at a specified frequency (Hz). Studies that did not meet these criteria were rejected on initial review. Studies that categorized subjects based on hearing ability or studied the use of hearing aids or implants were excluded from this study using the PRISMA 2020 flow protocol for new systematic reviews of searches conducted in only databases and registers . The following information was extracted from each study if available: author, year published, criteria defining subjects with AD, total number of subjects with AD, sex ratio of subjects with AD, mean age with standard deviation, pure-tone audiogram frequency and/or range of frequencies and associated hearing threshold linked to ear tested, and neurocognitive testing scores. Additional metrics were extracted , but due to limited reporting they could not be further analyzed. Due to variation related to the ear used for pure-tone audiometry , mean thresholds from the “better ear” were used. These data were also collected for the control cohort of each study. Data reported as a threshold frequency (Hz) for a specific pure-tone audiogram frequency or pure-tone audiogram frequency range was extracted directly onto an Excel® spreadsheet. For studies that reported this information using a 2D graph, WebPlotDigitizer© 4.4 was used to extract thresholds (Hz) from the y-axis to their corresponding frequency from the x-axis. All graphs were uniquely calibrated using to their pre-existing scale without correction. Pure tone audiometry thresholds were averaged for 500–1,000–2,000 Hz (referred to here as 0.5–2 kHz PTA) and separately for 500–1,000–2,000–4,000 (referred to here as 0.5–4 kHz PTA).Studies were assessed for potential bias using the Newcastle-Ottawa criteria for cohort studies by one rater (SSK)  between studies reflected differences in the reliability of the outcome measurements and not differences in outcome variability in the study populations. By doing this, studies with a small SD are given relatively higher weight while studies with larger SD are given relatively smaller weights. The weight given to each of the 6 studies was determined by the inverse-variance method to assign a quantitative value to how much influence each study has on the overall results of the meta-analysis. Inverse-variance is determined by taking the inverse of the variance of the effect estimate for each study . Therefore, more weight is given to studies with more precision compared to those with lower precision. Subgroup analysis was conducted for frequency ranges of 500–1,000–2,000 Hz (0.5–2 kHz PTA) and 500–1,000–2,000–4,000 Hz (0.5–4 kHz PTA). Differences in means between the AD and control groups were conducted using a two-tailed p < 0.001). The demographic characteristics for each study included in the meta-analysis is summarized in A total of six studies, totaling 171  subjects with AD and 222  control subjects , were included in data analysis. Not all studies reported neurocognitive testing outcomes. For subjects with AD, only four studies reported mean Mini-Mental State Examination (MMSE) data, and these scores were combined as the summary MMSE score. For the control cohort, only three studies reported mean MMSE data which were averaged as the collective MMSE score. Average MMSE score for AD subjects = 19 ± 4.3 (SD), and average MMSE score for control subjects = 27 ± 2.1 was significantly higher (p = 0.292).Due to the known positive correlation between age and hearing-loss . Five studies reported three frequency pure tone audiometry calculated for air conduction thresholds at 500, 1,000, and 2,000 Hz (0.5–2 kHz PTA). The meta-analysis of their means showed that the AD cohort had a 2.3 decibel hearing level (dB HL) higher compared to the control cohort (p < 0.002). p < 0.001).Six studies reported pure tone audiometry calculated for air conduction thresholds at 500, 1,000, 2,000, and 4,000 Hz (0.5–4 kHz PTA). The meta-analysis of their means showed that the AD cohort had a 4.5 decibel hearing level (dB HL) higher compared to the control cohort  and 0.5–4 kHz (4.5 dB difference), in subjects with AD compared to similar-aged controls. We found the difference in hearing thresholds at these two frequency ranges to be statistically significant where hearing threshold measured at 0.5–4 kHz PTA was greater by 2.66 dB than thresholds measured using 0.5–2 kHz PTA. These data suggest that AD is associated with an up to 4.5 dB hearing loss in frequencies associated with normal speech communication and that the loss of hearing is increased at higher frequencies.The methodology of this study was limited having a sole searcher (SSK) to identify potential studies to be included in the meta-analysis. Although the search methods were repeated by two other independent raters for verification (XMTN and DDW), having an initial search conducted by one individual may have introduced selection bias to this study. This meta-analysis was further limited by the small number of studies that met eligibility criteria. Several studies were excluded as they did not characterize AD specifically. In addition, few studies quantified peripheral hearing ability using pure tone audiometry thresholds at specific frequencies. Third, included studies were heterogeneous in their methods and reported outcomes that limited analysis to a small number of shared outcomes. Important subject characteristics such as highest level of education received, pre-retirement occupation, race, ethnicity, socio-economic status, medications taken, number and type of co-morbid conditions, number of years from initial diagnosis, and many other factors that could account for differences between groups was not reported in the majority of studies and therefore could not be analyzed in our meta-analysis. Moreover, history of noise exposure was not reported in any of the 6 studies used for this analysis. However, all 6 studies excluded subjects on the basis of hearing disorder diagnosis and deafness. Additionally, included studies varied in their definition of AD and their control population . However, despite these limitations which would be expected to dilute the impact of AD on hearing loss, we observed a statistically-significant increase in hearing threshold in AD subjects. Due to the aforementioned heterogeneity of reported results and lack of reported results in each individual study included in this analysis, it is possible that variables other than age and dementia impacted the difference in hearing ability of subjects with AD vs. controls. Although we sought to identify the potential for publication bias using the Newcastle Ottawa scale, we acknowledge that each study included in this meta-analysis featured an individual positive correlation between hearing loss and AD, which combined provided a pooled positive correlation. We cannot ignore the propensity of studies to report only positive findings, and therefore, we acknowledge that our meta-analysis may not have captured studies with negative findings on the relationship between hearing loss and AD, as no such studies were found in our search.There were 11 studies that were excluded from this meta-analysis due to the criteria established in the Methods section  was observed in the AD cohort relative to control. Although the clinical significance of this magnitude of difference is not yet known, it is important to note that this degree of hearing loss roughly corresponds to the thresholds for detecting amplitude modulations in sounds (Scheider and Pichora-Fuller, To our knowledge, this is the first meta-analysis providing quantification of peripheral hearing loss measured by pure tone audiometry for subjects with AD compared to age-matched controls. Our findings suggest that subjects with AD have higher hearing thresholds at 0.5–2 kHz PTA and 0.5–4 kHz PTA compared to age-matched controls. This finding is supported by current literature from epidemiological studies on the relation of ARHL and AD. Our meta-analysis suggests that in future studies peripheral hearing should be better characterized in AD cohorts compared to age-matched controls accurately estimate the contributions of peripheral hearing loss to cognitive impairment. In addition, to use peripheral hearing loss in AD as a modifiable risk factor, assessing hearing ability using pure-tone audiometry on a routine basis would be critical. Additionally, analyses comparing pure tone audiometry measurements to other characteristics associated with AD may yield improved understanding on the effects of peripheral hearing in AD and elucidate effects of confounding variables that could not be analyzed in this study. Lastly, to characterize the pathophysiologic relationship between age-related hearing loss and AD, future studies should utilize pure-tone audiometry alongside other audiologic and neurophysiologic measures of peripheral and central hearing.Publicly available datasets were analyzed in this study. All data are publically available in the publications cited in the article.SK performed the systematic literature review, determined study inclusion/exclusion, extracted and analyzed data, and prepared the manuscript. X-MN validated study methods, performed data analysis, prepared the manuscript, and edited the manuscript. DW validated study methods, prepared figures, and edited the manuscript. RM determined study inclusion/exclusion criteria, validated extracted data, and prepared and edited the manuscript. DL determined study inclusion/exclusion, validated study methods, validated extracted data, and prepared and edited the manuscript. All authors contributed to the article and approved the submitted version.RM was supported by an ASH Foundation New Century Scholars Research Grant. DL was supported by an award from the Benjamin R. and Elinor W. Bullock and Edwin E. and Jeanne Bullock Goldberg Professorial Scholar Fund.DL has consulted for Eisai Pharmaceuticals in the past year. The remaining 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.