There was tremendous increase in the number of cases of mucormycosis among patients affected by coronavirus disease 2019 (COVID-19) during the second wave of pandemic in South Asian countries. This invasive fungal infection primarily affects paranasal sinuses and can have orbito-facial and intracranial extension. We are presenting the radiological findings of invasive mucormycosis with pathological and clinical outcome correlation. It is important for radiologists to have the knowledge of various presentations of this opportunistic infection for early diagnosis and helping clinicians in planning the appropriate line of management. The study also emphasizes on the correlation between the extent of involvement with clinical outcome and we proposed a magnetic resonance imaging (MRI) based scoring system to standardize and prognosticate the patients affected with mucormycosis.Materials and methods: We utilized GE 1.5 tesla, 16-channeled MRI machine for scanning the clinically suspected mucormycosis patients and did plain and contrast study of the paranasal sinuses, orbito-facial study and included brain as and when required. Images were acquired in axial, coronal, and sagittal planes using T1, T2, and fat-saturated short tau inversion recovery sequences (STIR), fat-saturated contrast sequences for better evaluation of the extent of the disease. Diffusion-weighted sequence was also acquired to detect ischemic changes in optic nerve or brain parenchyma. Contrast study was used to detect any major vessel occlusion or cavernous sinus thrombosis in the study population.Results: Total number of cases (n) included in the study were 32. The mean age group was 41-50 years with the median age was 47 years. Out of 32 cases (n=32), in 16 cases (50%) the disease was limited only to the paranasal sinuses and in remaining 16 (50%) cases, disease has spread to other regions such as orbits, facial soft tissues, optic nerve, and brain parenchyma. All the 18 cases with Mild score (MRI ROCM score 1-3) survived and all those with severe score (2 cases) (MRI ROCM score 7-10) did not survive.Conclusion: During the second wave of COVID-19 pandemic, we observed a signiﬁcant rise in acute invasive mucormycosis infection primarily involving the paranasal sinuses and spread to orbito-facial, cerebral parenchyma causing related complications and hence increased morbidity and death. Radiologically, using MRI, it was effectively possible to detect early extrasinonasal spread and other fatal complications thereby guiding the physicians and surgeons in the proper early aggressive management of the disease. Here, we have described the radiological characteristics of paranasal sinus mucormycosis and its spread to other regions. We also proposed an MRI-based Scoring System for standardized assessment of the disease severity. We observed in our study that the extent of disease on MRI is directly correlating with mortality. Mucormycosis (previously known as zygomycosis) is a serious but rare fungal infection caused by a bunch of molds known as mucormycetes. These molds live throughout the surroundings. This angioinvasive fungal infection commonly affects individuals with low immunity, elderly especially chronic diabetics, and individuals with chronic steroid or iatrogenic immunosuppression and patients with hematological malignancies, and those who have undergone organ transplantation use . It mostDuring the second wave of the coronavirus disease 2019 (COVID-19) pandemic in India, there was an increased incidence of mucormycosis infection in COVID-19 patients due to various factors. Rampant steroid use, broad-spectrum antibiotics use, diabetes, use of unsterile oxygen masks/ventilators, and lack of awareness/hygiene could have contributed to the increased incidence.This invasive fungal infection most often primarily affects paranasal sinuses then spreads to orbits, brain, and facial skeleton and causes orbital cellulitis, optic neuritis, encephalitis, or infarction. It is more aggressive with rapid progression and may also show recurrence in cases of incomplete surgical debridement or improper medical management. It may also cause death due to complications such as intracranial hemorrhage. Mortality rates due to intraorbital and intracranial complications of mucormycosis range from 50% to 80% . Hence, The clinical symptoms depend on the site of involvement and common presenting symptoms which include nasal blockade, proptosis, facial pain and edema, ptosis, chemosis, anesthesia/decreased sensation over affected face, and even ophthalmoplegia. In cases of intracranial extension, headache, altered sensorium, fever, and various other neurological signs and symptoms can be noted ,5.The study is conducted in the Department of Radiology, Malla Reddy Narayana Multispeciality Hospital, Suraram, Hyderabad. Here we present magnetic resonance imaging (MRI) findings of paranasal sinuses, orbital and cerebral mucormycosis. Follow-up of these cases for clinical outcome and correlation with biopsy was done to the extent possible.The methods available to detect or diagnose mucormycosis infection are multidetector computed tomography (MDCT), MRI, laboratory analysis such as biopsy, and microscopic analysis. MDCT and MRI are non-invasive and key imaging modalities available not only for diagnosis but also for detecting disease extension and its complications thereby significantly helping in guiding proper management.We used GE 1.5 tesla, 16-channeled magnetic resonance imaging (MRI) machine  for scanning patients, referred for suspected mucormycosis, and did both plain and contrast study of paranasal sinuses, orbito-facial study, and included brain as and when required. Images were acquired in axial, coronal, and sagittal planes using T1, T2, and fat-saturated short tau inversion recovery sequences (STIR), and fat-saturated contrast sequences for better evaluation of site and extent of disease. Diffusion-weighted sequence was also acquired to detect ischemic changes in optic nerve or brain parenchyma. Contrast study helped to detect cavernous sinus thrombosis in our study.Inclusion and exclusion criteriaWe included both COVID-19 and post-COVID-19 patients having clinical suspicion of mucormycosis fungal infection and referred for paranasal sinus, brain, and orbit imaging in the period between April 2021 and June 2021. Cases outside the study period were not included, including non-COVID-19 patients.Our study includes a total of 32 cases (n=32) out of which the clinical outcome of 23 cases was followed up while the clinical outcome of nine cases could not be followed up. Most common age group in our study was 41-50 years and the 21-30 years age group had the least number of cases /STIR sequence, MRI is superior to CT in detecting subtle extrasinonasal soft tissue extension like early infiltration into brain or orbit, and also optic nerve involvement. Marrow edema is also better appreciated in MRI much before evident on CT as bone lysis/destruction.In this scoring system we included paranasal sinus involvement, extrasinonasal spread, such as to the orbit, maxillofacial soft tissue, optic nerve, brain, and bone involvement assigned the score as depicted in Table Highest individual score has been assigned to intracranial extension as it majorly influences the severity and clinical outcome. Our scoring system has a maximum score of "10" which is simple, fast tracking, and easier to implement in emergency situations like the recent COVID-19-associated mucormycosis surge. Based on our proposed MRI-based scoring system, 24 75%) cases were mild, six (18.8%) cases were moderate, and two (6.3%) cases were severe in our study population (n=32)  showed significant clinical improvement and the severe form (score 7-10) succumbed to the disease Table .Mucormycosis spreads through the nasal route, often the nasal or paranasal sinuses are the primary sites of involvement thereby spreading to the para-antral soft tissues, facial bones, base of skull, and central nervous system. In addition to diffuse alveolar injury with severe inflammatory exudation, COVID-19 patients have immunosuppression due to decrease in CD4+ and CD8+ T cells .Multiple reasons were postulated for the increase in incidence of mucormycosis in patients with COVID-19 during the second wave of the COVID-19 pandemic in India. The reasons mentioned in the previous literature are as follows: (1) immunosuppression caused by the COVID-19 infection itself which poses additional risk in patients with other comorbidities, (2) hyperferritinemic syndrome which is seen in severe COVID-19 disease that poses a risk for cellular damage and increased free iron that ultimately increases risk for acquiring mucormycosis ,8, (3) hSpread to orbit is thought to be via the nasolacrimal duct and erosion of medial orbital wall, congenital dehiscence in medial wall of orbit, and also through the arterial or venous perforators in the medial wall of orbit. Such invasion is facilitated by the thinness of the lamina papyracea, congenital dehiscence often present along the medial wall, and the perforations of the medial wall by arteries and veins ,10.Mucormycosis is an angioinvasive fungus, its spread to brain is thought to occur from angioinvasion causing occlusion/thrombosis of vessels leading to cerebral infarction and dissemination to central nervous system -13. SpreThe presenting symptoms vary depending on the site of involvement but most often fever, pain and facial swelling, blurring of vision, cranial nerve palsies from cavernous sinus involvement, and even loss of vision or paraparesis from cerebral infarction would be the presenting symptoms. However, it is a fatal fungal disease with rhino-cerebral manifestations being the most common .Cavernous sinus thrombosis is well known vascular complication of this fungus and occurs from the orbit, across the orbital apex, and on MRI the cavernous sinus thrombosis appears as loss of flow void on T1- and T2-weighted imaging and absent enhancement on contrast images .Mycotic aneurysms are another form of vascular complications of mucormycosis in addition to thrombo-occlusion . VasculaAll patients suspected of rhino-cerebro-ocular mucormycosis received antifungal medication and underwent magnetic resonance imaging of paranasal sinuses (MRI-PNS) examinations. Few patients were primarily referred for computed tomography of paranasal sinuses (CT-PNS) followed by an MRI-PNS. Based on the MRI findings, endoscopy and biopsy were suggested.All the suspected patients received different treatments and surgical procedures depending on the involvement and extension of the pathology. Few patients underwent endoscopic debridement of devitalized fungal infected tissue, few underwent inferior maxillectomy, while few underwent facial resection. Few patients with partial visual loss underwent orbital debridement.Diagnostic tests available for confirmation are KOH (10-20% potassium hydroxide) staining, microscopy, histopathology of debrided tissue and culture, Grocott’s methenamine silver stain, and periodic acid Schiff (PAS) staining.The study was conducted during the second wave of the COVID-19 pandemic, where there was increased prevalence of concomitant mucormycosis cases. So, the study emphasized on mucormycosis infection. However, few bacterial infections like Klebsiella can also cause similar aggressive necrotizing bacterial rhinitis with different pathological and microbiological profiles and these bacterial infections are usually sporadic in nature .The following images demonstrate different cases of rhino-orbito mucormycosis with varied patterns of sinonasal involvement and extrasinonasal spread such as optic nerve involvement Figures -1F, intrWhite et al. conducted a study including 135 adults with COVID-19 infection and observed an incidence of 26.7% invasive fungal infections . Song etMetwally et al. in their study published in 2022 used a diagnostic-reliable staging method to define the imaging features of post-COVID-19 head and neck mucormycosis and identify risk variables for extrasinus extension. They proposed an MR imaging-based classification of mucormycosis staging depending on the site and extent of involvement into stage 1 to stage 4, stage 1 is limited to sinonasal, stage 2 is extrasinus maxillofacial, stage 3 is intraconal and stage 4 is intracranial involvement. The prevalence of mucormycosis in stages 1, 2, 3, and 4 in our study was 11.1%, 55.6%, 20.6%, and 12.7%, respectively .MRI imaging characteristics of rhino-orbtio-cranial mucormycosisFeatures of sinusitis include mucosal thickening/opacification/fluid levels, frothy secretions, pan sinus involvement, and on MRI mucosal thickening appear T1-hypo and T2-hyper or -hypointense; fluid secretions appear as T2/STIR hyperintense. Intrasinus fungal elements may appear as low signal intensity on T2 sequences. Intrasinus fungal ball/mycetoma appears as irregular, spherical/mass-like intrasinus variable T1/T2 signal with convex margin often nonenhancing. Extrasinonasal soft tissue involvement appears hyperintense on FAT-SAT/STIR images and shows mild enhancement on contrast images.Bone InvolvementAffected bone marrow appears hypointense on T1W and hyperintense on STIR images with heterogeneous post-enhancement .Intracranial ExtensionInvolved areas appear T1-hypointense, T2/STIR hyperintense, and many times show diffusion restriction in cases of cerebral infarction by vessel occlusion and appear T1-hypointense, T2/FLAIR hyperintense, and may or may not show diffusion restriction.Intraorbital ExtensionEspecially on STIR, hyperintensity in intraconal soft tissue, T2/STIR hyperintensity in pre-septal/post-septal orbital cellulitis, altered signal in optic nerve/perioptic region, optic nerve appear thickened, show diffusion restriction, and enhancement in post-contrast images in case of optic nerve involvement.Contrast EnhancementThree patterns are recognized in sinus involvement which are homogenous, patchy heterogeneous, and non-enhancing tissue. Non-enhancing tissue represents necrosed tissue, filling defect in cavernous sinus in cases of cavernous sinus invasion or thrombosis and occlusion of major vessels in cases of cerebral infarction.Black turbinate sign, though not specific to mucormycosis, is also often found in rhino-nasal mucormycosis. Non-enhancement of turbinate (commonly middle or inferior) on contrast image suggesting necrosed tissue referred to as black turbinate sign which appears on endoscopy as black eschar, a sentinel sign of rhino-orbito-cerebral mucormycosis ,26. ThisLimitationsSince the study included a smaller population, further large population studies are invited to collect more data in the future so as to standardize the reporting system and to further validate the proposed MRI-based ROCM scoring system.In our study, we observed the rampant raise of mucormycosis cases primarily involving the paranasal sinuses then contiguously spreading to the orbit, face, and brain causing devastating complications likely optic neuritis, encephalitis, brain infarctions, and even death.With the use of available imaging information, we described the different types of MRI imaging characteristics of rhino-orbito-cranial mucormycosis and postulated the MRI-based scoring system to unify and standardize the reporting system and the study needs more data in the future for recommendation and validation.


Caenorhabditis elegans avoidance of the mild and medium hyperosmolality of 0.41 and 0.88 Osm but does not affect avoidance of high osmolality of 1.37 and 2.29 Osm. The interaction between ASH and ADL is actualized through a negative feedback circuit consisting of ASH, ADL, and RIM interneurons. In this circuit, hyperosmolality-sensitive ADL augments the ASH hyperosmotic response and animal hyperosmotic avoidance; RIM inhibits ADL and is excited by ASH; thus, ASH exciting RIM reduces ADL augmenting ASH. The neuronal signal integration modality in the circuit is disexcitation. In addition, ASH promotes hyperosmotic avoidance through ASH/RIC/AIY feedforward circuit. Finally, we find that in addition to ASH and ADL, multiple sensory neurons are involved in hyperosmotic sensation and avoidance behavior.Sensations, especially nociception, are tightly controlled and regulated by the central and peripheral nervous systems. Osmotic sensation and related physiological and behavioral reactions are essential for animal well-being and survival. In this study, we find that interaction between secondary nociceptive ADL and primary nociceptive ASH neurons upregulates  Animals regularly encounter, sense, and avoid noxious environmental stimuli, such as hyperosmotic and hypoosmotic stress , excessivia a relay of a peripheral inhibitory neuron as proposed by gate control theory , and volatile repellents  channel and triggers hyperosmotic avoidance  interneurons, and ADL. The circuit osmolality-dependently and differently regulates C. elegans avoidance of mild, medium, and high osmolality. It upregulates and does not affect medium and high osmolality avoidance, respectively. Upon hyperosmotic stimulation, both the primary nociceptor ASH and secondary nociceptor ADL are excited; the excited ADL excites ASH by the signaling pathway of FLP (FMRF-Like Peptide)-4 and the NPR (NeuroPeptide Receptor)-5 receptor; RIM interneuron, is excited by ASH through glutamate/NMR (NMDA class glutamate Receptor)-2 signaling, inhibits ADL via the tyramine/TYRA (TYRAmine Receptor)-3 pathway. Thus, ASH exciting RIM reduces ADL exciting ASH. The neural signal integration modality in the circuit is disexcitation. The circuit functions to fine-tune hyperosmotic sensation and avoidance. Disexcitation is a newly found modality that establishes the homeostasis of pumping and 5-HT production in food-sensing ADFs under food supply and deprivation conditions ::TeTx transgenic animals were positively related to the osmolality by glycerol/M13 solutions of varied concentrations (50) 0.8614 and 0.9775 Osm in the WT and ADL::TeTx animals, respectively. The ADL neurotransmission elimination significantly reduced animal avoidance of mild and medium hyperosmolality of 0.41 and 0.88 Osm by 0.1 and 0.5 M glycerol/M13 solutions. However, it did not affect the avoidance of high osmolality of 1.37 and 2.29 Osm by 1 M or 2 M glycerol/M13 solutions, compared with its effect in the WT N2. The difference was most significant at 0.88 Osm. We thus used 0.5 M glycerol/M13 solution (0.88 Osm) for the following experiments in this study unless otherwise indicated. We further used 0.25 M NaCl/M13 (0.77 Osm), 0.5 M fructose/M13 (0.87 Osm), and 0.5 M sorbitol/M13 (0.90 Osm) solutions to validate the observed behavioral response to 0.8 Osm glycerol/M13 is hyperosmotic avoidance. As expected, WT N2 animal displayed similar hyperosmotic avoidance of these solutions . HisCl1 is a histamine-gated chloride channel subunit from istamine . Our resvoidance , support2+) responses to the medium hyperosmolality of 0.88 Osm by 0.5 M glycerol/M13 solution, using fluorescent Ca2+ imaging with GCaMP3.0 as a Ca2+ indicator  and unc-31(e928) mutant animals to assay the cell autonomy of the ADL hyperosmotic responses. Unc-13 (UNCoordinated) encodes syntaxin-1 binding protein UNC-13, which is required for synaptic vesicle fusion with the presynaptic membrane and thus essential for neurotransmitter release , which is essential for the exocytosis of dense cored vesicles and thus the release of neuropeptides  animal  animal ,G. The rTeTx, ADL::TeTx, and ASH/ADL::TeTx. The expression of TeTx in ASH and ADL was directed by promoters srv-11p (in ASH), ver-2p , and gap-11p , respectively. Interestingly, genetic inhibition of neurotransmission in ADL, ASH, and ADL/ASH neurons similarly reduced the hyperosmotic avoidance of 0.88 Osm hyperosmolality by glycerol/M13 solution in transgenic animals  to identify the role of neuropeptide signaling in ADL regulating ASH. The egl-3 gene encodes a homolog of mammalian proprotein convertase EGL (EGg Laying defective)-3 essential for neuropeptides biosynthesis , and ADL::egl-3(RNAi) genetic manipulations on ASH Ca2+ responses to the 0.88 Osm stimulation. ADL::TeTx, ADL::unc-31(RNAi), and ADL::egl-3(RNAi) similarly decreased ASH Ca2+ responses to the hyperosmolality  mutant showed augmented hyperosmotic avoidance. However, the behavioral phenotype could not be restored to the WT by the reconstitution of flp-21 genomic DNA driven by its promoter of 4.1 kb flp-21p  mutant animals of npr-4(tm1782) and npr-5(ok1583) showed significantly decreased hyperosmotic avoidance compared to WT N2 , npr-5(ok1583), and double knockout (DKo) of flp-4(sy1606); npr-5(ok1583), displayed almost similar defects in hyperosmotic avoidance. Only double gene rescued animal of ADL::flp-4; ASH::npr-5; DKo restored WT behavior; both single gene rescue animals of ADL::flp-4; DKo and ASH::npr-5; DKo phenotypically copied animals of single flp-4 and npr-5 mutant and the double mutant  and npr-5(ok1583) animals showed similar decreased ASH Ca2+ responses to the stimulation of 0.88 Osm glycerol/M13 solution. In contrast, ADL::flp-4; flp-4 and ASH::npr-5; npr-5 rescue animals showed WT Ca2+ responses -1, NLP-3, and NLP-15, for neurotransmission  displayed augmented hyperosmotic avoidance that contradicts the expected phenotype. In addition, the behavioral phenotype was not restored to the WT in transgenic animals expressing flp-21 in ASH/ADL and ASH/ASI  animals were less sensitive to medium hyperosmolality of 0.88 Osm. Although eat-4 rescue expression in its expression cells driven by its promoter eat-4p (5.6 kb) restored WT hyperosmotic avoidance in the transgenic animal, eat-4 re-expression in ASH/ASI and ASH/ADL showed no effect on the behavior ::eat-4(RNAi), ASH/ADL::eat-4(RNAi), and ASH/ASI::eat-4(RNAi) showed a significantly increased avoidance ratio , ASJ (Amphid Single Cilium J), ADF , AFD (Amphid Finger-like Endings D), and AWB (Amphid Wing Neuron B), reduced hyperosmotic avoidance  and nmr-2(tm3785) displayed notably augmented hyperosmotic avoidance. The reconstitution of nmr-2 in its expression cells driven by its promoter nmr-2p (4.9 kb) restored the WT behavioral phenotype  nmr-2 rescue and TeTx expression restored the WT behavior and displayed augmented hyperosmotic avoidance, respectively  animal significantly decreased. The signals were fully restored to the WT by the extrachromosomal re-expression of eat-4 cDNA in ASH, using two cell-specific promoters, gpa-11p (in ASH and ADL) and sra-6p (in ASH and ASI) with an expression overlap is ASH. However, reconstitution of eat-4 in ADL, which is postsynaptic to RIM, did not rescue the RIM Ca2+ responses to 0.88 Osm osmolality (2+ responses in the nmr-2(ok3324) significantly decreased and restored to the WT in the RIM-specific nmr-2 rescued animal  animal displayed significantly augmented avoidance of 0.88 Osm glycerol/M13 solution. This behavioral phenotype was fully restored to the WT by tdc-1 genetic rescue expression in its expression neurons (RIM and RIC) and RIM alone, driven by tdc-1 promoter (3.0 kb) and gcy-13p (2.3 kb), and by administration of exogenous tyramine (5 mM). Furthermore, the RIM-specific tdc-1 knockdown showed the same behavioral effect as the tdc-1 mutation -55. We thus used the receptor mutant animals to test which receptor/s function/s in animal hyperosmotic avoidance. Among ser-2, tyra-2, tyra-3, and lgc-55 mutant animals, only the tyra-3(ok325) showed non-WT behavior, a significantly augmented hyperosmotic avoidance /CEP /PDE (Posterior DEirid) and AIM (Anterior Interneuron M)/AFD . Thus, RIM modulates ADL activities, possibly through tyramine/TYRA-3 signaling. Tyramine is a monoamine neuromodulator . It is smutation . This revoidance . The hyp2+ responses to 0.88 Osm glycerol/M13. ADL Ca2+ signals in tdc-1(n3419) and RIM::TeTx neurotransmission-eliminated animals were augmented similarly. The increased Ca2+ response in the mutant animal was restored to the WT by the RIM-specific tdc-1 genetic rescue and the treatment with 50 μM exogenous tyramine (2+ responses to 0.88 Osm glycerol/M13 should change similarly in tyra-3(ok325) and tdc-1(n3419) animals. As expected, the ADL Ca2+ signals in the tyra-3(ok325) significantly increased and were restored to the WT by ADL::tyra-3 genetic rescue but not by the application of exogenous tyramine  and that generates increased aversion behavior , AVB , AVD , and AVE . ASH connects with interneurons controlling or modulating locomotion by chemical and electric synapses , inx-4(e1128), and ASH::TeTx, and were restored to the WT in transgenic animals of the RIC::unc-9;unc-9 and ASH::inx-4; RIC::inx-4; inx-4  mutant displayed reduced hyperosmotic avoidance; the RIC-specific tbh-1 rescued and exogenous octopamine-treated tbh-1(n3247) animals showed the WT behavioral phenotype , SER (SERotonin/octopamine receptor)-3, and SER-6. We next employed receptor mutant animals to identify the octopamine receptor/s involved in hyperosmotic avoidance. Ser-6 and octr-1 mutant animals displayed significantly reduced hyperosmotic avoidance. The behavioral defects were eliminated by the gene rescue driven by their promoters  animal and restored to the WT in the RIC-specific gene rescued animal  and RIC-specific ser-6 rescued animals  and RIC::TeTx animals and restored to the WT in the RIC-specifically tbh-1 rescued animal  and high hyperosmolality . The differentiated roles of intensity-or concentration-dependent sensations in animal behaviors, which display widely in the animal species, are intrinsic traits essential for animal adaptation to environmental changes. In addition to the ADL case, there are the following instances. The concentrations-dependent sensations of isoamyl alcohol mediated by ODR-3 in AWC responding to only lower concentrations of the odorant and ASH responding to only higher concentrations of odorant induce odor attraction and avoidance behaviors  displays increased avoidance of the medium hyperosmolality, and the behavioral phenotype cannot be restored to the wild type by the flp-21 genetic rescue in its expression cells or ADL alone. FLP-21 is a known ligand of the NPR-1 receptor. The function of FLP-21 should be discussed in conjunction with its receptor, NPR-1. NPR-1 signaling in ASH inhibits C. elegans’ avoidance of copper ions and hyperosmolality by glycerol under food deprivation (flp-18 and flp-21 results in increased swimming rates (flp-21(ok889) that was unable to be rescued by flp-21 reexpression is that only one strain was used in this study. In addition, mutation of other gene/s in these mutant animals should not be excluded. The background gene mutation may also explain varied dauer entry in C. elegans strains of flp-21(ok889), flp-21(pk1601), and flp-21(sy880) display, among them, only flp-21(pk1601) showed non-WT phenotype  downregulates by suppressing ADL hyperosmotic response through TYRA-3 signaling, avoidance of mild and medium osmolality in C. elegans. Interestingly, in addition to the action on AIY, octopamine functions as an autocrine signal to auto-excite via SER-6 signaling.Octopamine and tyramine play antagonistic roles in regulating multiple physiological processes and behaviors. In behavior . In the behavior . This st2+ responses to 3 M fructose  genes: gcy-25, gcy-32, gcy-33, gcy-34, gcy-35, gcy-36, and gcy-37. Both neurons may sense hyperosmolality by guanylyl cyclases—cGMP—TAX-2/TAX-4 signaling. However, molecular mechanisms of hyperosmotic sense in these neurons need further study. Among the known and potential hyperosmolality-sensitive neurons, ASH, ADL, ASE, ASG, and AWC express osm -9 encoding a homolog of the mammalian TRPV channel OSM-9, which acts in the detection of strong external osmotic shocks. These sensory neurons have chemo-or/and electro-synaptic connections with interneurons or/and motor neurons, such as AVA, AVB, AVD, AVE, AIA, AIB, AIZ, and RIA. These neurons control or regulate animals’ locomotion  plates at 20°C using E. coli bacteria OP50 as food by standard procedures  were recombined into the pDEST™ R4-R3 Vector II or custom-modified destination vectors using attL-attR (LR) recombination reactions to generate expression clones.All expression constructs were generated with a Three-Fragment Multisite Gateway® system . Briefly, three entry clones comprising three PCR products . The PCR products of promoters were amplified from C. elegans genomic DNA with primers containing 15–20 bp sequences that carry some sequence of attL4 and attR1 recombination sites. Alternatively, promoter vectors in the C. elegans Promoters Library  were directly used. The length of each promoter used in this study is as follows: ver-2p 2.7 kb (in ADL), gpa-11p 3.3 kb (in ASH and ADL), sra-6p 3.8 kb (in ASH and ASI), srv-11p 1.9 kb (in ASH), flp-21p 6.6 kb, npr-5p 3.0 kb, npr-4p 4.0 kb, eat-4p 5.6 kb, sra-9p 4 kb (in ASK), flp-17p 3.3 kb (in BAG), srh-11p 0.7 kb (in ASJ), srh-142p 3.5 kb (in ADF), gcy-32p 0.8 kb , str-1p 4.0 kb (in AWB), str-2p 3.7 kb (in AWC), srsx-3p 0.9 kb (in AWB and AWC), gcy-5p 3.2 kb (in ASER), gcy-7p 1.3 kb (in ASEL), gcy-15p 0.8 kb (in ASG), nmr-2p 4.9 kb, gcy-13p 2.3 kb (in RIM), tdc-1p 3.0 kb (in RIM and RIC), tyra-3p 4.2 kb, dat-1p 0.7 kb , gcy-18p 0.8 kb (in AFD and AIM), tbh-1p 4.5 kb (in RIC), ser-6p 3.5 kb, octr-1p 3.9 kb, and ttx-3p 3.1 kb . The following genes were used to create the B entry clones. The flp-21, npr-4, npr-5, tdc-1, octr-1, and ser-6 genes were amplified from the genomic DNA of wild-type N2 worms. Nmr-2, eat-4, tyra-3, ser-6, and tbh-1 cDNA were amplified by reverse transcription-PCR (RT-PCR) from C. elegans mixed-stage RNA. Their PCR products flanked by attB1 and attB2 were recombined with the pDONR-221 vector containing attP1 and attP2. To generate entry clone C, the BP reaction sites attB2r and attB3 were inserted into the sequences of the sl2e::TagRFP-t, sl2d::GFP, or unc-54 3’ UTR and recombined with the PDONR-P2R-P3 vector. All primers for cloning these promoters and genes are listed in BP recombination reactions were used to generate entry clones B (containing a sequence of a tested gene) and C (containing Hyperosmotic solutions were prepared by putting solutes in the M13 buffer. M13 buffer consists of (in mM) Tris 30, NaCl 100, and KCl 10 . The solutions’ osmolality was measured using Osmomat-3,000 . The measured osmolality (in Osm) was: M13 buffer, 0.28; 0.1 M glycerol/M13 0.41; 0.5 M glycerol/M13 0.88; 1 M glycerol/M13 1.37; 2 M glycerol/M13 2.29; 0.25 M NaCl/M13 0.77; 0.5 M fructose/M13 0.87; 0.5 M sorbitol/M13, 0.90.via a glass micropipette onto the tail of the forward-moving worm. All tested animals’ avoidance response to the droplet of hyperosmotic solutions was scored as the percentage of animals that displayed reversal or Ω turn for more than one-half of body length within 4 s.All experiments were performed using synchronized young adult animals maintained on 6 cm nematode growth medium (NGM) plates and at 20°C. The hyperosmotic shock test was performed using a wet-drop test for assaying avoidance of acute hyperosmotic simulation, as previously described . BrieflyA droplet assay was used to examine the response to gradual internal osmolality upshifts, essentially as described . Shortly2+ indicators G-CaMP3 or G-CaMP6F. The calcium indicators were excited by 460–470 nm light emitted by an Osram Diamond Dragon LBW5AP light-emitting diode (LED) model  constructed in a multi-LED light source  and filtered with a Semrock FF01-520/35–25 emission filter . Fluorescence images were captured with an Andor iXonEM + DU885K EMCCD camera with 256 × 256 pixels at 10 frames per second under an Olympus IX-70 inverted microscope  equipped with a 40× objective lens [numerical aperture (NA) = 1.3]. A homemade PDMS microfluidic device was used to trap worms and deliver solutions  of the tested neuron soma was captured and analyzed using Image-Pro Plus 6.0 . A nearby region with an area similar to that of the tested soma was used to measure background signals. The percent change of average fluorescence intensity 0ΔF/F was plotted as a function of time for all curves. Where ΔF = F﹣0F; F, the average fluorescence intensity of the region of interest (ROI) of neuronal soma in each frame; 0F, the average fluorescence intensity of the ROI within the initial 5 s  or 60 s (in RIM) before application of the hyperosmotic solution. The average background signal was subtracted from F and 0F. The Ca2+ signals were shown by curves, box plots, and heat maps. Box plots showed analyzed data of the average intensity of Ca2+ signals during glycerol/M13 solution perfusion.We employed Day-1 adult animals for all calcium imaging and used them once. Animals collected from culture plates were put into the M13 buffer to remove bacteria for 10 min before being loaded into the microfluidic chip. Because Drosophila HisCl1 gene in the tested neurons and employed 10 mM histamine to activate the channels. For the preparation of test agar plates containing 10 mM histamine, a stock solution of histamine (1 M in M13 buffer) was diluted with ultrapure water into a 10 mM working solution, and 10% (v/v) working solution was added to the agar solution at approximately 60°C before making the plates. Tested animals were transferred to the test plate containing 10 mM histamine or no histamine as a control and let them freely move for 10 min before hyperosmotic shock assay. HisCl1 transgenic animals were pretreated for 10 min in M13 buffer without (as controls) or with 10 mM histamine for calcium imaging experiments. HisCl1 channel may have a low probability of opening in the absence of ligand histamine, causing a leaking current in some cases. To reduce the leaky effect on tests, we reduced the injected plasmids containing HisCl1.For chemogenetic silencing of the tested neurons, we used neuron type-specific promoters to drive specific expression of the TeTx to intercept vesicular release. TeTx is a specific protease of synaptobrevin that has been successfully used to block vesicle fusion with presynaptic fusion, thus inhibiting or eliminating chemical or even electrical synaptic transmission of tested neurons in C. elegans . When the comparison was limited to 2 groups, an unpaired t-test was used to analyze differences and calculate p-values. When more than two groups of data were compared, data were analyzed by ordinary one-way or two-way analysis of variance (ANOVA), with recommended post hoc tests in the GraphPad Prism 8 software package. Dunnett’s multiple comparison correction was applied when multiple samples were compared to a single sample, i.e., wild-type N2 or other controls. Tukey’s multiple comparison correction was used when multiple samples were compared. The p-value is indicated as follows: ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and in different colors for varied comparisons.Data of avoidance ratio are displayed as box plots, with each dot representing the data from each individual tested animal or each test. The CaThe original contributions presented in the study are included in the article/Z-XW supervised the study. HL, J-JW, and RL performed the major part of the experiments, analyzed the data, and created the figures. P-ZW, J-HH, YX, P-PW, J-LZ, and S-JL performed the minor part of the experiments. HL and Z-XW wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China (32071013 and 31471034).The authors declare that the research was conducted without 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.


We attempted to explore the association between metformin use and hearing loss in in a large-scale study. This retrospective multicenter cohort study assessed the data of patients with type 2 diabetes mellitus (DM) aged over 40 years using the Observational Health Data Science and Informatics open-source software and the Common Data Model database from 1 January 2002 to 31 December 2019. Each participant was selected using the ICD-10-CM diagnosis code E11 for type 2 DM with sensorineural hearing loss. The participants were divided into metformin and non-metformin users. The outcome measure was the first occurrence of hearing loss after the diagnosis of DM as measured by the CDM cohort study. A total of 80,596 patients, including 46,152 metformin users and 34,444 non-metformin users from three hospitals were assessed. After calibration, we compared the risk of hearing loss using Kaplan–Meier curves, and found significant differences between the groups. The calibrated hazard ratio in the three hospitals  was summarized. These findings suggest that the probability of hearing loss-free survival in the metformin user group is higher than that in the non-metformin user group. Hearing loss is defined as the partial or total inability to hear. It can be mild, moderate, or severe, and can affect one or both ears. Currently, approximately 20% of the global population lives with hearing loss . HearingThe impact of hearing loss is broad and can be catastrophic. It includes the loss of the ability to communicate, which can lead to social isolation. Assistive technologies, including hearing aids, cochlear implants, closed captioning, and other devices, can help with hearing loss . HoweverNoise-induced and age-related hearing losses result from cochlear hair cell injuries that occur through mechanical and metabolic injuries . CommonlMetformin, which is commonly administered to patients with diabetes for its antidiabetic effect, is known to have an antioxidant function that blocks the cell apoptosis cycle related to ROS and reduces the oxidative stress levels in rats ,8. SeverWe aimed to validate the relationship between metformin use and hearing loss by using a large-scale common data model (CDM); this relationship has been confirmed in animal experiments and retrospective human studies ,11. The This retrospective study of Korean patients who visited the ANAM, GURO, or ANSAN hospital between 1 January 2002 and 31 December 2019 used Observational Health Data Sciences and Informatics (OHDSI)  open-souAll patients were aged over 40 years, had type 2 DM, and were identified using the ICD-10-CM diagnosis code E11. These are identified by the concept IDs (identifiers) in the CDM database, the details of which are provided in The primary outcome of this study was the first occurrence of hearing loss after the diagnosis of type 2 DM. Hearing loss corresponds to sensorineural, sensory, neural, noise-induced, and sudden sensorineural hearing loss, which are mapped to the systematic nomenclature of medicine used as the vocabulary for diagnosis codes in We defined potential confounders based on age, sex, history of hyperlipidemia, hypertension, cardiovascular disease, and use of aspirin, nonsteroidal anti-inflammatory drugs, and statins .2 value test [http://www.R-project.org accessed on 10 October 2020) and the OHDSI Cohort Method R package [This study utilized preference scores, which were obtained by transforming propensity scores (PSs), to measure the relative preference for metformin and non-metformin users in relation to patient demographics and health characteristics. This approach yields scores that are more readily interpretable. The equation for preference scores is as follows: ln((preference score)/(1 − preference score)) = ln((PS)/(1 − PS))-ln(proportion/(1 − proportion)), where “proportion” represents the proportion of participants receiving the treatment . To contlue test . Statist package . The metThe total number of patients from the three hospitals included in the study was 80,596 .The 1000 person-year incidence rate (IR) of hearing loss in metformin users (IR = 3.64) was slightly higher than that in non-metformin users (IR = 3.58). ANAM hospital showed the same trend (IR = 3.70 to 3.61), whereas the GURO (IR = 3.28 to 3.66) and ANSAN (IR = 4.41 to 4.82) hospitals demonstrated an opposite trend. These results indicated that the time at risk in the metformin group was longer than that in the non-metformin group. In addition, the median follow-up times were 2470.5, 2524.5, and 2062 days for the ANAM, GURO, and ANSAN hospitals, respectively. The follow-up distribution plots are provided in Preference scores are a transformation of PSs adjusted for differences in group size.The selected baseline characteristics of metformin and non-metformin users from all hospitals before and after PS matching and stratification are described in The current study presents Kaplan–Meier curves for the cumulative risk of hearing loss in patients with diabetes who were metformin or non-metformin users. PS calibration was performed with 10 strata and hospitals . The comThe probability of hearing loss-free survival in metformin users was higher than that in non-metformin users.Hearing loss causes difficulty to patients, and they often seek effective treatments; therefore, a drug that could decrease hearing loss would be a great discovery. Among the candidate drugs is metformin, which is known and proven to be safe and is already prescribed to patients with DM worldwide. The mechanism of action of metformin in cochlear hair cells has only been confirmed in animal models through several studies ,11; howeBased on the data of a large patient group in three hospitals, the groups were homogenized and analyzed according to metformin use. Variations in the patient group were corrected through covariate balancing, PS matching, and stratification. PS indicates the probability that a particular patient is assigned to a treatment group according to the given covariates. These probabilities are calculated using a logistic regression, and each patient has a PS. When we observed differences in the covariates between two groups, we calculated the standard mean difference (SMD). The SMD represents the similarity of the distribution of covariates of both groups. This is known as PS matching.In this study, the IR of hearing loss for each hospital was higher in the metformin user group, with the exception of the group from the ANSAN hospital. This finding should be considered with exposure time, which is the time at risk of DM; therefore, the metformin users showed a lower incidence rate than non-metformin users. In the demographics of each group, the prevalence of hypertension and cardiovascular disease was higher in metformin users before PS matching and stratification. However, no significant difference was found between the two groups after correction with PS matching and stratification. Furthermore, no significant differences were noted between other potential confounders.We used a random-effects model to assume the effect size considering the difference in each hospital, whereas the fixed-effects model considered each hospital identically. The most evident difference between the two methods was the variation control. While fixed-effects models are used only within variations, random-effects models are used between variations.The mechanism underlying the association between metformin use and its risk-reducing effect on hearing loss has not been fully elucidated. In vitro research has suggested that metformin is a potential agent against ototoxicity that reduces the number of ROS produced by all processes . In our Therefore, future randomized prospective studies are warranted to determine the protective effects of metformin on hearing loss in humans. DM medications have the same mechanisms of action as metformin; therefore, disentangling their associations from those of metformin could confirm and redeem the previous results. In addition, in vivo studies are needed to identify and understand the direct mechanism of action of metformin on ear cells. This research will serve as a bridge for the development of innovative therapeutics for the prevention of hearing loss in the future.This cohort study using CDM data found that among patients with DM, those who used metformin had a lower risk of hearing loss than those who did not use metformin. These findings suggest an association between hearing loss and metformin use. This pioneering study suggests a new aspect for hearing loss, for which a prominent preventive method has yet to be identified. Several prior experiments in animal models support this finding; however, confirming this in a large-scale patient group by a retrospective cohort study based on the CDM method is necessary. Further research on the mechanism underlying this association and the possible prophylactic methods in patients with hearing loss by complementing these limitations is warranted.


Hearing loss impacts the quality of life and affects communication resulting in social isolation and reduced well-being. Despite its impact on society and economy, no therapies for age-related hearing loss are available so far. Loss of mechanosensory hair cells of the cochlea is a common event of hearing loss in humans. Studies performed in birds demonstrating that they can be replaced following the proliferation and transdifferentiation of supporting cells, strongly pointed out on HCs regeneration as the main focus of research aimed at hearing regeneration. Neurotrophins are growth factors involved in neuronal survival, development, differentiation, and plasticity. NGF has been involved in the interplay between auditory receptors and efferent innervation in the cochlea during development. During embryo development, both NGF and its receptors are highly expressed in the inner ears. It has been reported that NGF is implicated in the differentiation of auditory gangliar and hair cells. Thus, it has been proposed that NGF administration can decrease neuronal damage and prevent hearing loss. The main obstacle to the development of hearing impairment therapy is that efficient means of delivery for selected drugs to the cochlea are missing. Herein, in this study NGF was administered by the intranasal route. The first part of the study was focused on a biodistribution study, which showed the effective delivery in the cochlea; while the second part was focused on analyzing the potential therapeutic effect of NGF in senescence-accelerated prone strain 8 mice. Interestingly, intranasal administration of NGF resulted protective in counteracting hearing impairment in SAMP8 mice, ameliorating hearing performances  and hair cells morphology . The results obtained were encouraging indicating that the neurotrophin NGF was efficiently delivered to the inner ear and that it was effective in counteracting hearing loss. Hearing loss is a common sensory disorder characterized by sensorineural hearing loss generally starting in individuals over 65 years. It impacts the quality of life and affects communication resulting in social isolation and reduced well-being , 2. The In the past two decades, an important piece of studies has been devoted to hearing research and to the comprehension of biological mechanisms contributing to hearing loss . HearingWhile the cellular and molecular mechanisms of ARHL are poorly understood, preclinical and clinical studies reported that loss of synapses between inner hair cells (IHCs) and SGNs, also known as cochlear or IHC synaptopathy, is an early pathology that precedes IHC loss in mice  and humaLoss of mechanosensory HCs of the cochlea is a common event of hearing loss in humans . StudiesDuring embryo development, both NGF and its receptors are highly expressed in the inner ears. It has been reported that NGF is implicated in the differentiation of auditory gangliar and HCs . Thus, iIn this work, the effect of NGF has been studied in an animal model of ARHL, using the senescence-accelerated prone strain 8 (SAMP8) mice. SAMP8 mice are a strain derived from AKR/J mice, chosen for a phenotype toward either accelerated senescence with a mean life span of 9 months . It has The main obstacle to the development of hearing impairment therapy is that efficient means of delivery for selected drugs to the cochlea  are missTherefore, the local route to the inner ear may overcome the toxic effects due to the systemic drug administration allowing drug repurposing for hearing loss. The possibility of a minimally invasive alternative route of administration may have the potential to transform not only the therapeutic approaches of hearing impairment but also the related unmet health demands of tinnitus and balance disturbs as that intravitreal administration has revolutionized the treatment of retinal disorders. To address this issue, herein, NGF was administered by the intranasal route, and in the first part of the study a biodistribution study was performed in C57BL/6, showing the effective delivery in the cochlea. In the second part of the study, the therapeutic potential of intranasal administration of NGF was evaluated. Notably, the neurotrophin was able to counteract ARHL, ameliorating hearing performances and morphological changes. The results obtained were encouraging indicating that the neurotrophin NGF was efficiently delivered to the inner ear and thus proposing NGF as an efficient effector for the protection and recovery of hearing loss.n = 19). Mice were housed in macrolon cages with filter hoods, in a room where the air was continuously filtered, thereby avoiding contamination. All experiments were performed in the compliance with the European Directives . Animals arrived on site 4 days before compound administration to allow optimal acclimation. During experiments, paired animals were caged at a controlled temperature with a day/night cycle of 12/12 h. Animals received water (control tap water) and nutrition ad libitum. Animal were purchased from Envigo, USA. Animals were randomly distributed over the different experimental conditions and blinding was applied during data acquisition and analysis when possible.For this study as strain, C57BL/6 males were selected (n = 3), (2) NGF treated and sampling at 2 h (n = 4), (3) NGF-treated and sampling at 12 h (n = 4), (4) NGF compound treated and sampling at 24 h (n = 4), (5) NGF compound treated and sampling at 48 h (n = 4)  vehicle that received only saline buffer and sampling at 12 h . Standard references were diluted at 1000, 500, 250, 125, 62.5, 31.25, 15.63, and 0 pg/mL to perform the standard curve. The Standard working solution at various concentrations was added into two wells and the samples were to other wells. The plate was sealed and incubated for 1 h and 30 min at 37 °C. Then the liquid of each well was aspirated, and immediately 100 μL of Biotinylated Detection solution was put into each well. The plate was again sealed and incubated for 1 h at 37 °C. After incubation, the solution from each well was removed and 350 μL of wash buffer was added. After extensive washes, 100 μL of HRP-conjugated working solution was added to each well. The plate was again sealed and incubated for 30 min at 37 °C. After washes, 90 μL of Substrate Reagent was put in each well and incubated for 15 min at 37 °C. Finally, 50 μL of Stop Solution was added to each well and read at microplate reader at 450 nm.n = 24). All experiments were performed in the compliance with the European Directives . Mice were housed in macrolon cages with filter hoods, in a room where the air is continuously filtered, thereby avoiding contamination. Animals were randomly distributed over the different experimental conditions and blinding was applied during data acquisition and analysis when possible. Mice were divided in Group 1: SAMR1 (negative control n = 8), Group 2: SAMP8 + vehicle (n = 8) and, Group 3: SAMP8 + NGF (n = 8). NGF was daily administered by intranasal route (10 µL)  male 1-month-old were purchased from Envigo n = 4. All exABRs are electric potentials recorded from scalp electrodes, and the first ABR wave correspond to the total activity of the auditory nerve fibers communicating the IHCs. For the procedure, animals were anesthetized using a ketamine/xylazine mixture, and body temperature was maintained at 37 °C using a heating pad. Earphones were positioned in the left ear of each mouse, an active electrode was positioned in the vertex of the skull, a reference electrode under the skin of the mastoid bone, and a ground electrode in the neck skin. The stimuli involved tone pips of five frequencies (2–24 kHz) at various sound levels (from 0 to 90 dB) varying to cover the mouse auditory frequency range. ABR analysis of each mouse were executed individually and using the OtoPhyLab system. Evoked potentials were obtained by the signal averaging technique for each noise level and ABR thresholds for each frequency were revealed using OtoPhyLab software.DPOAEs are acoustic signals produced and amplified by the cochlear epithelium, suggesting an index of cochlear functionality. They are linked to OHCs health which amplifies sound-evoked cochlear vibrations. They are independent from IHCs or auditory nerve fibers. For the procedure, animals were anesthetized using a ketamine/xylazine mixture, and a probe (OtoPhyLab) was introduced into the external left ear canal. The primary tone F2 was set at five frequencies (4–kHz) at 58 dB. The frequency ratio F2/F1 was set at 1.2. At all frequencies (F2), the input of DPOAE systems was received, digitized, and calculated using the output of the microphone. The amplitudes of the frequency component at the distortion product frequency were established and represented.g at 2–8 °C. The supernatant (plasma) was kept at −80 °C until analysis. Prestin quantification for each animal was performed by the ELISA method .For each animal, 2 mL of blood was collected by cardiac puncture and put in a tube containing EDTA and immediately centrifuged for 15 min at 1000 × Left cochleae of all mice were extracted and fixed with paraformaldehyde solution overnight and then, decalcified for 7 days in EDTA. The membranous and sensory spiral enclosing the organ of Corti were isolated and the HCs were immunolabeled for anti-Myosin-VIIa . The medial region of the OC was mounted on glass slides and observed using a confocal microscope. The total number of IHCs and OHCs was counted for the mid-segment. For the ribbon synapse immunostaining, the right cochleae of 1 animal/group were sampled, fixed, and decalcified as previously stated, and the ribbon synapses of the mid-segment were immunolabeled for anti-GluR2  and the cochlear neurons using anti-Tuj1 . Samples were mounted on glass slides and observed using a confocal microscope. Processing and analysis of immunohistochemistry images were performed using ImageJ with Fiji. The number of ribbon synapses immunostained for GluR2 and was counted using the Analyze Particles Plugin.2 2 mM) overnight at RT. The stria vascularis, tectorial, and Reissner’s membranes were separated by microdissection. After washing in PHEM buffer, samples were dehydrated in a graded series of ethanol (30–100%), critical point dried in CO2, coated with gold palladium, and examined using a scanning electron microscope Hitachi S4000.Right cochleae of 3 animals/group were extracted from the temporal bone and fixed in 2.5% glutaraldehyde in PHEM buffer . A No mortality and pathological clinical signs were observed between groups at the analyzed time points. As expected, a significant reduction in body weight was observed in the SAMP8+vehicle group compared to the SAMR1 control group from day 33  in the mid-turn of the cochlea. OHCs loss was confirmed in the SAMP8 animals treated with vehicle  channels open, leading to the entry of potassium from the potassium-rich endolymph, depolarizing the HCs to stimulate the production of the neurotransmitter glutamate at their ribbon synapses . The orgThe ribbon synapses connecting IHCs and SGN afferent fibers are susceptible to ototoxic agents, aging, and noise damage. This has been observed in in vivo models of noise-induced impairment in which a favored and irreversible loss of a subpopulation of IHC afferent synapses, those responsible for the so-called high threshold, low spontaneous activity fibers , 34 has Injury to ribbon synapses was initially identified as “hidden hearing loss,” a damage of hearing quality appearing in presence of a normal audiometric hearing thresholds. emerging studies in the field suggests that this alteration in neuronal connectivity is at basis of auditory problems, distinguished by a failure to hear sounds such as a conversation, in the presence of background noise , 34.Similar to the BBB, BLB defends the cochlea and vestibular system causing limiting the drug to reach the inner ear upon systemic administration . This haDespite promising results obtained with drug intratympanic administration, it appears as an invasive method and leakage of liquid preparations through the Eustachian tube is the most important limit of this route. Thus, finding an alternative route of administration that effectively deliver the drug and is minimally invasive is challenging.Indeed, the main strength of this study is that we selected the intranasal route of administration and that it was effective in delivering NGF in the target area (patent No. EP22188990.0 (05-08-2022).This choice is derived from the analysis of previous reports demonstrating that the perilymph in the tympanic scale derives from cerebrospinal fluid (CSF) and possesses the same composition. Moreover, it is known that the perilymph in the scala vestibuli comes from blood plasma across a blood-perylimph barrier, whereas that of the scala tympani originates from CSF. In fact, the cerebrospinal fluid–perilymph barrier comprises the cochlear aqueduct , which jOur results provide the first demonstration that NGF, reaching the cochlea, exerted significative protective effects in preserving HCs and number, as well as ribbon synapses and Glutamate receptors, thus indicating that the intranasal administration is a less invasive and more efficient administration route.In this study, SAMP8 mice were selected as a model to detect the otoprotective effect of NGF and, notably, the neurotrophin could preserve the function of OHCs safeguarding hearing loss in aging mice. Specifically, electrophysiology studies reported a protective effect in animals treated with NGF both at the functional and morphological levels. In this condition, NGF efficiently decreased click-evoked ABR threshold shifts compared to vehicle-treated ears. Interestingly, SAMP8 mice treated with NGF once a day for two months by intranasal route presented a significant decrease in ABR threshold, an increase of DPOAE amplitude, and a decrease of plasma prestin and cochlear HC loss at 3 months old compared to the vehicle group, paralleled by the protection of HCs, particularly of OHCs, as highlighted by cytocochleogram. Moreover, the histological characterization by SEM and immunohistochemistry of cochleae demonstrated the absence of stereocilia fusion of IHCs and an increase of the number of ribbon synapses and cochlear neurons after the compound treatment corroborating electrophysiology, otoacoustic and functional data. These results are in agreement with previous observations on DBA/J2 mice treated with mouse NGF, where an antiapoptotic and pro-survival role has been proposed for this neurotrophin .Our results provide for the first time, evidence that the intranasal administration route efficiently targets the cochlea promoting the maintenance of IHC, OHC, and ribbon synapses and their functionality during aging, supporting the potential of NGF administration as a strong therapeutic approach to reduce the burden of ARHL also in humans.Taken together, these results confirm that the NGF performs significant protective efficacy on age-related hearing impairment by ameliorating hearing performances and preserving histopathology of the cochlea in the preclinical model of accelerated senescence SAMP8.Supplementary figureAJ checklist


Mucormycosis is a potentially fatal fungal infection, and there is limited information on its precise epidemiology and treatment practices, including the optimal dosage of liposomal amphotericin B.A retrospective, multicenter, nationwide analysis of 82 proven and probable cases of mucormycosis was performed. Cases between 2015 and 2022 were collected from 51 hospitals in Japan by hematologists and infectious disease specialists. The study included the epidemiology, treatment details, and association between the dose of liposomal amphotericin B and the outcome.Rhizopus spp., Cunninghamella spp., and Mucor spp. were the most common organisms. Mortality at 4 weeks was 41.5%. The survivors had a shorter duration of neutropenia (P = .006) and less persistent hyperglycemia (P = .023). The site of infection and species of Mucorales had no detectable effect on survival. Survival did not differ between patients receiving liposomal amphotericin B at 5 mg/kg/d relative to those receiving >5 mg/kg/d (P = .625). Using Cox proportional hazards models and adjusting for confounders, the hazard ratio for the influence of >5 mg/kg/d liposomal amphotericin B on 4-week survival was 0.86  compared with 5 mg/kg/d.The lungs were the most commonly involved organ (70.7% of cases), and 35.4% of patients had disseminated disease. This study provides important insights into the precise epidemiology and treatment practices of mucormycosis. Treatment with liposomal amphotericin B at doses higher than 5 mg/kg/d did not improve outcomes relative to 5 mg/kg/d. Mucormycosis is a rare but potentially fatal fungal infection that poses significant diagnostic and treatment challenges . IndividCurrent treatment options for mucormycosis include surgical resection and antifungal therapy . AlthougThe aim of this nationwide observational study was to provide accurate and comprehensive epidemiologic and therapeutic information about mucormycosis in Japan. In addition, we sought to determine if administration of liposomal amphotericin B at >5 mg/kg/d to patients with mucormycosis was associated with improved outcomes. Thus, we aimed to determine the optimal dosing regimen for liposomal amphotericin B that improves therapeutic outcomes while minimizing adverse effects.The Japanese Society for Medical Mycology conducted this retrospective, multicenter, nationwide study in collaboration with the Japanese Society of Hematology and the Japanese Association for Infectious Diseases. Patient data were collected from training institutions of the Japanese Society of Hematology, certified institutions of the Japanese Association of Infectious Diseases, and members of the Japanese Society for Medical Mycology working in hospitals with >100 beds. Between January 1, 2015, and June 30, 2022, the charts of all patients in these hospitals who had been diagnosed with mucormycosis by culture, polymerase chain reaction, or pathology were analyzed. Survival data were obtained by collecting information on cases 120 days after the specimen collection date on which the first diagnosis of mucormycosis was based. Patient data were collected anonymously from each institution. The Nagasaki University Hospital Clinical Research Ethics Committee approved this study .Mucorales genera, age, sex, underlying medical conditions, surgical interventions, and antifungal therapy. Infection sites were categorized by organ, and disseminated infections were defined as infection present in ≥2 organs. Risk factors for invasive fungal infections, including neutropenia and hematologic malignancy status, and various immunosuppressive medications for underlying diseases, such as corticosteroids, T-cell immunosuppressants, or B-cell immunosuppressants, were also evaluated [Data on the various parameters were collected from individuals diagnosed with mucormycosis, including geographic regions in Japan, year of diagnosis, the rationale behind the diagnosis, site of infection, valuated . Prolongvaluated , 14–19.Mucorales genera, characteristics of mucormycosis patients, and characteristics of mucormycosis treatment. In addition, the geographical distribution of mucormycosis cases and Mucorales genera in Japan was determined. In this study, the date at which the initial specimen was collected for the diagnosis of mucormycosis was defined as day 0 for calculating the number of days of survival.Patients were classified into proven, probable, and out-of-diagnostic definition groups according to the European Organization for Research and Treatment of Cancer and Mycoses Study Group Education and Research Consortium (EORTC/MSGERC) consensus definitions of invasive fungal disease . EpidemiMucorales genera, age, sex, underlying disease, and neutrophil status. Treatment characteristics analyzed included timing of resection of infected lesions, antifungal agents already administered at the time of diagnosis, antifungal agents used to treat mucormycosis, and timing of initiation of liposomal amphotericin B. Outcomes analyzed were intolerance of liposomal amphotericin B leading to discontinuation, postmortem diagnosis, and probability of survival at 4, 6, and 12 weeks postdiagnosis.A comparative analysis of patients diagnosed with proven or probable mucormycosis who received liposomal amphotericin B maximum doses >5 mg/kg/d vs those who received maximum 5 mg/kg/d was performed to assess the prognostic association of liposomal amphotericin B dose on patient outcome. Patients whose maximum dose was <5 mg/kg/d or no liposomal amphotericin B were excluded, as were cases in which liposomal amphotericin B was administered for <7 days. Patient characteristics analyzed included the site of infection, causative 2 test. The association between liposomal amphotericin B dose and survival was evaluated using Kaplan-Meier statistics for the 5 mg/kg/d and >5 mg/kg/d liposomal amphotericin B groups. Differences were tested using the log-rank test. To control for confounding, a Cox proportional hazards model was performed, with the outcome being an interval from diagnosis to death of ≤4 weeks. The treatment variable was the maximum dose of liposomal amphotericin B (5 mg/kg/d or >5 mg/kg/d). Confounders controlled for in this analysis were age (>65 years), no recovery to >1000 neutrophils by the end of observation, and resection of the infected lesions, which clinically appear to have a clear prognostic association with the outcome of mucormycosis [P value <.05 was considered statistically significant.Observed characteristics related to mucormycosis were summarized using appropriate descriptive statistics. Differences in all categorical variables were tested using the Fisher exact test or χrmycosis , 20, 21.Mucorales genera (P = .457) , as shown in We performed an analysis of 82 cases of mucormycosis from 51 institutions in Japan . Our res = .457) . Further = .457) . The mosP = .880) (P = .006), persistent hyperglycemia (P = .023), and a higher incidence of the need for intensive care (P < .001)  , but the < .001) . HoweverP < .001) (P = .244). The maximum dose of liposomal amphotericin B used varied from <5 mg/kg/d to >10 mg/kg/d, but there was no difference in the doses administered between survivors and nonsurvivors (P = .570). Of the 64 patients treated with liposomal amphotericin B, 15 (23.4%) required dose escalation due to poor response to treatment, and 6 (9.4%) discontinued treatment due to intolerance to liposomal amphotericin B. The median duration of treatment with liposomal amphotericin B ≥5 mg/kg/d in the survival group (interquartile range) was 36.5 (0–70) days. The timing of initiation of liposomal amphotericin B at a dose of ≥5 mg/kg/d was not significantly different between survivors and nonsurvivors (P = .237)  . Several = .237) . TreatmeMortality rates at each time point were 41.5% (34/82) at 4 weeks, 45.1% (37/82) at 6 weeks, and 52.4% (43/82) at 12 weeks after diagnosis. The number of patients diagnosed at postmortem examination was 9 (10.8%). No prognostic differences were observed based on specific organ involvement .Mucorales genera, or patient background except for brain infection, which occurred only in the patients who received >5 mg/kg/d  (P = .796) compared with 5 mg/kg/d, and no superiority of >5 mg/kg/d over 5 mg/kg/d was demonstrated  was compared with that of 15 patients who were treated with a dose >5 mg/kg/d (>5 mg/kg/d group) . No sign mg/kg/d . There w mg/kg/d . Survivactively) . Using Cnstrated .This study elucidated the precise epidemiology and treatment outcomes of patients with mucormycosis across the adult and pediatric spectrum. The strength of this study lies in the analysis of mucormycosis cases with strict and precise definitions, as shown in This study showed that the mortality rate for mucormycosis managed by hematologists and infectious disease specialists, regardless of the organ involved, remains substantial at 41.5% at 4 weeks after diagnosis. Furthermore, the stable mortality rate from 2015 to the present suggests a lack of improvement in treatment strategies for mucormycosis. Contrary to previous reports, the prognosis of mucormycosis treated by hematologists and infectious disease specialists did not vary significantly by organ involved . This fiRhizopus spp. was the most common causative microorganism of mucormycosis, as in other countries [Cunninghamella spp. was as common as Mucor spp. It has been reported that Cunninghamella spp. are frequently isolated from pulmonary and disseminated lesions [Cunninghamella spp. than previously reported. It has also been reported that mucormycosis caused by Cunninghamella spp. has a higher mortality rate than infections caused by other genera [Cunninghamella spp. were more likely to have pulmonary or disseminated mucormycosis and were more likely to die because of their complex clinical history. This study also yielded interesting results regarding the importance of blood cultures in mucormycosis. Our results showed that 9.8% of mucormycosis patients managed by hematologists and infectious disease specialists had positive blood cultures, particularly those with Mucor spp. Adding blood culture to the diagnostic protocol for mucormycosis patients with host factors and Mucor spp. may be beneficial, particularly in the case of disseminated mucormycosis.Our study also shed light on the epidemiology of mucormycosis in Japan. Although there has been a worldwide increase in the incidence of COVID-19-associated mucormycosis , 22, thiountries , 9, 15;  lesions . Our focr genera , 6, althOne of the epidemiologic features of mucormycosis in Japan is the low prevalence of hyperglycemia (14.6%) compared with reports from other countries. The prevalence of diabetes mellitus as an underlying disease in patients with mucormycosis varies widely in different regions of the world. It ranges from 44% to 75% in India and Iran, 52% in the United States, 27% in Australia, and 17% to 23% in Europe . Thus, iTreatment-related findings were observed, with surgical resection being frequently used in the survival group. While the design of the present study precludes a definitive determination of causality between surgery and prognosis, it remains a pivotal observation that the 4-week survival rate in patients without surgery was only 38.9%, in stark contrast to the 95% survival rate demonstrated in patients who underwent surgical resection within 3 days of diagnosis. Combination of liposomal amphotericin B with other antifungal agents did not show a detectable synergistic effect. There was no significant difference in the timing of initiation of treatment with ≥5 mg/kg/d of liposomal amphotericin B between the survivors and nonsurvivors.Our study evaluated the clinical outcomes of liposomal amphotericin B administered at different doses for mucormycosis. We found no significant differences in outcome between the 5 mg/kg/d and >5 mg/kg/d groups. Even after adjustment for some limited confounders, no survival benefit was observed with doses >5 mg/kg/d. The results support the use of 5 mg/kg/d as an appropriate starting dose in the treatment of mucormycosis, although it should be noted that this study cannot exclude the possibility that >5 mg/kg/d was used in patients with more severe disease. Clinicians would benefit from evidence supporting the use of 5 mg/kg/d because very high doses  have been associated with increased nephrotoxicity and hypokalemia . BecauseThe limitations of this study should be considered when interpreting the results. First, mild cases of mucormycosis with primarily superficial lesions may have been underrepresented because data were not collected from specialists such as otolaryngologists and dermatologists. Therefore, the epidemiologic and treatment outcomes of this study may only represent the characteristics of mucormycosis seen by hematologists and infectious disease specialists. Second, nonresponse bias may exist because not all hospitals in Japan were studied. Nevertheless, responses were obtained from 317 institutions—representing 37.8% of the total 838 target institutions, consisting of training institutions of the Japanese Society of Hematology, certified institutions of the Japanese Association for Infectious Diseases, and some of the members of the Japanese Society for Medical Mycology—and thus were likely to provide accurate epidemiologic data. Third, the impact of isavuconazole on mucormycosis was not considered in this study because isavuconazole was approved in Japan in December 2022. Fourth, the end point in our study did not include nephrotoxicity. At a higher dose of 10 mg/kg/d, it has been reported that 40% of patients experienced dose reduction or discontinuation due to nephrotoxicity , making In conclusion, we have successfully elucidated the precise epidemiology and treatment outcomes of mucormycosis managed by hematologists and infectious disease specialists in Japan. In this study, we found that there was no difference in the outcome of mucormycosis in patients who received liposomal amphotericin B at 5 mg/kg/d vs >5 mg/kg/d. As the evidence for the treatment of mucormycosis is limited mainly to observational and retrospective studies, additional clinical trials are needed to improve the prognosis of this disease. We believe that the results of our study will provide insight into the management of current mucormycosis cases and provide a foundation for future research.ofad480_Supplementary_DataClick here for additional data file.


Rheumatoid arthritis (RA) can affect the auditory system either as a direct complication of the disease course or secondary to medication adverse effects. Rheumatoid arthritis-induced autoimmune inner ear disease can present as tinnitus, conductive hearing loss, sensorineural hearing loss (SNHL), or mixed. According to previously published articles, SNHL is the most common hearing loss in RA. Age, smoking, noise exposure, and alcohol may affect the disease progression. Here, we present a case of a 79-year-old female who presented to the rheumatology clinic with complaints of abrupt onset bilateral hearing loss with associated tinnitus; pure tone audiometry confirmed sensorineural hearing loss. Her tinnitus resolved completely, and her hearing improved significantly after treatment with steroids and leflunomide. Based on this case and previous literature, we conclude that rheumatoid arthritis is the cause of SNHL in our patient. Appropriate and timely medical interventions have been reported to improve the prognosis of hearing impairment in rheumatoid arthritis patients. Our case highlights the need to have a high index of suspicion of rheumatoid arthritis-induced autoimmune inner ear disease in an elderly patient presenting with sudden-onset hearing impairment and the importance of prompt referral to a rheumatologist. Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that affects multiple organs such as the heart, lungs, skin, and eye in addition to primary joint manifestations . The audThe pathophysiology of hearing impairment in RA is not well known. However, some findings show relations to arthritis of incudostapedial and incudomalleolar joints resulting in conductive hearing loss or deposition of the immune complex, hence sensorineural hearing loss . Other eDiagnosis is based on clinical findings, and different types of hearing tests are available; pure tone audiometry (PTA) from 125 to 8,000 Hz is mostly used for evaluating hearing loss (HL) .Treatment for RA-induced autoimmune inner ear disease is high-dose glucocorticoids (60 to 80 mg prednisone) every morning for two to three weeks and commencement or escalation of disease-modifying antirheumatic drugs (DMARDs) . This usThe patient is a 79-year-old female with a past medical history of normal pressure hydrocephalus , nicotine dependence, and 40 years history of meningioma. She was referred by the otolaryngologist to our rheumatology clinic after being diagnosed with SNHL and positive rheumatoid factor (RF). Four months before her rheumatology clinic presentation, the patient developed sudden-onset unilateral hearing loss. She stated that she woke up one morning and could not hear from her right ear. Within one week, her symptoms progressed to bilaterally hearing loss with associated tinnitus. She was evaluated by an otolaryngologist and underwent pure tone audiometry  of the head without contrast revealed a 2.6 cm left frontal lobe meningioma with associated local mass effect, a right transparietal approach ventriculostomy shunt, and persistent ventriculomegaly (stable chronic findings compared to her previous head imaging)  revealed no acute intracranial abnormality and no enhancing retrocochlear mass lesion Figures , 4.At the rheumatology clinic, the patient complained of bilateral hearing loss, tinnitus, and joint pains in her hands and feet. She also reported morning stiffness in her hands lasting less than 30 minutes daily. She denied joint swelling, ear pain, discharge, recent head trauma, nasal congestion, sore throat, fever, and chills. She takes no medication. Upon chart review by the rheumatologist, it was noted that the patient was diagnosed with rheumatoid arthritis at an outside facility 11 years before the index presentation. At that time (11 years ago), she complained of bilateral ankle pain, and her investigation was remarkable for +RF IgM > 100, +RF IgG 7.5, +RF IgA 20, +CCP >300, and erythrocyte sedimentation rate (ESR) 17 and C-reactive protein (CRP) 0.5. Computed tomography of the ankle was reported as osteoarthritis (OA). There was no referral letter to a rheumatologist. Laboratory findings at the outside facility 11 years before the index presentation are shown in Table Physical examination of her auricular cartilages, ear canals, and tympanic membranes showed no erythema, swelling, or tenderness. Ear canals were patent and intact. The whisper test revealed significantly reduced hearing in the left ear and no hearing in the right ear; normal oral aperture; no oral ulcerations and no skin ulcerations or pits on fingertips; and squaring over the bilateral carpometacarpal joint, no joint tenderness, erythema, and swelling. Anterior/posterior view of the hand X-ray revealed significant joint space narrowing and findings of both rheumatoid arthritis and osteoarthritis Figure . LaboratThe patient received counseling on smoking cessation and was started on prednisone 60 mg daily. Her hearing impairment significantly improved after four weeks of steroid therapy. Prednisone was gradually tapered, and she was started on a steroid-sparing agent/DMARD, leflunomide.Rheumatoid arthritis (RA) is a chronic inflammatory condition that primarily affects the synovium causing destructive arthritis . HoweverA pathologic condition that damages the cochlea and/or cochlear nerve results in sensorineural hearing loss (SNHL) . SeveralAs an autoimmune condition, RA is linked to adaptive immune system malfunction, which leads to the development of various autoantibodies ,23. The SNHL in RA could also be caused by retrocochlear involvement because immunological complexes that are formed in the endothelium of the vasa vasorum, which supplies the cochlear nerve, may cause auditory neuropathy . MedicatLifestyle and environmental factors are synergistically associated with SNHL in patients with RA. Elderly patients with RA are known to be prone to hearing impairment , and nicOur patient thought her symptoms were due to her history of meningioma and normal pressure hydrocephalus; however, magnetic resonance imaging of her brain showed no enhancing retrocochlear mass lesion. Her symptoms improved following the commencement of prednisone medication, suggesting that the cause could have been most likely autoantibody damage to the hair cells or from immune complex deposition on the cochlear hair cells. This case highlights the need to have a high index of suspicion of rheumatoid arthritis-induced autoimmune inner ear disease in elderly patients presenting with sudden-onset hearing impairment and the importance of prompt referral to a rheumatologist.While it is primarily known that there are many potential causes and multiple contributors to the pathogenesis of sensorineural hearing loss in patients with rheumatoid arthritis, the temporal association of hearing impairment without other strong evidence favors rheumatoid arthritis as the culprit of SNHL in our patient. Using ototoxic medications for different purposes and the effects of DMARDs could be potential confounders; however, there was no history of prior use in our patient. We also highlight the variability in steroid response to autoimmune SNHL and the need to consider steroid-sparing agents as they could be beneficial. Several studies have shown improved outcomes with appropriate and timely effective medical intervention.