Newborn hearing screening has to be considered the first step of a program for the identification, diagnosis, treatment and habilitation/rehabilitation of children with hearing impairment.In Tuscany Region of Italy, the universal newborn hearing screening is mandatory since november 2007. The first guidelines for the execution of the screening have been released in June 2008; then many other Italian regions partially or totally adopted these guidelines. On the basis of the experience from 2008 and according to the recent evidences in the scientific literature, a new screening protocol was released in Tuscany region. The new protocol is an evolution of the previous one. Some issues reported in the previous protocol and in the Joint Committee on Infant Hearing statement published in 2007 were revised, such as the risk factors for auditory neuropathy and for late onset, progressive or acquired hearing loss. The new updated guidelines were submitted to the Sanitary Regional Council and then they have been approved in August 2016. The updated screening protocol is mainly aimed to identify newborns with a congenital moderate-to-profound hearing loss, but it also provides indications for the audiological follow-up of children with risk’s factor for progressive or late onset hearing loss; further it provides indications for the audiological surveillance of children at risk for acquired hearing impairment. Then, in the new guidelines the role of the family paediatrician in the newborn hearing screening and audiological follow-up and surveillance is underscored. Finally the new guidelines provide indications for the treatment with hearing aids and cochlear implant, in accordance with the recent Italian Health Technology Assessment (HTA) guidelines.In the paper we report the modality of execution of the universal newborn hearing screening in the Tuscany Region, according to the recently updated protocol. The main features of the protocol and the critical issues are discussed.The online version of this article (10.1186/s13052-017-0397-1) contains supplementary material, which is available to authorized users. The universal newborn hearing screening (NHS) is aimed to identify babies with moderate to profound hearing impairment present at birth  , 2.NHS has to be considered as a first step of a program for the identification, diagnosis, treatment and habilitation/rehabilitation of children with hearing impairment .2 and a population of about 3.8 million inhabitants (2016).Tuscany is a region in central Italy with an area of about 23,000 kmTotally, there are 26 childbirth points in Tuscany: 25 are public structures and 1 is in a private clinic. In 2015 488.000 babies were born in Italy, of which 27.500 in Tuscany .In Tuscany Region of Italy, the first guidelines for the execution of the Universal Newborn Hearing Screening (UNHS) have been released in June 2008; then many other Italian regions partially or totally adopted these guidelines. Furthermore, in the 2017 “essential health care levels”  document, the NHS is reported to be mandatory for each Italian region.In 2011 a report by the Regional Agency for Health on NHS execution in Tuscany attested that 100% of the Tuscanian childbirth clinics executed the UNHS and that more than 98% of the neonates born in Tuscany underwent the procedure [On the basis of the experience from 2008 and according to the recent evidences in the scientific literature, a new screening protocol was released in Tuscany region. The new protocol was based on the previous one and on the Joint Committee on Infant Hearing (JCIH) statement published in October 2007. In the JCIH statement, indeed, there is a list of all auditory risk factors marked with different symbols, in order to point out the indicators associated with an higher risk . All infants with indicator for hearing loss are reported to be referred for an audiological assessment at least once by 24–30 months of age; children with risk factors associated with an higher risk for delayed-onset hearing loss have to substain more frequent audiological assessments . These aThe updated screening protocol is mainly aimed to identify newborns with a congenital unilateral or bilateral, moderate-to-profound hearing loss, but it is also provides indications for the audiological follow-up of children with risk’s factor for progressive or late onset hearing loss; further it provides indications for the audiological surveillance of children at risk for acquired hearing impairment. Then, in the new guidelines we underlined the role of the family paediatrician in the newborn hearing screening and audiological follow-up and surveillance.Finally the new guidelines provide indications for the treatment with hearing aids (HA) and cochlear implant (CI), in accordance with the recent Italian Health Technology Assessment (HTA) guidelines .In the following sessions we report the modality of execution of the UNHS in the Tuscany Region in Italy, according to the recently updated protocol.In Tuscany, the centres that are involved in the NHS and in the early diagnosis and treatment of hearing impairment in childhood are divided in 3 levels, depending on facilities and personnel available in the childbirth and in the audiologic clinic. The modalities of screening execution are adjusted on the bases of each centre’s resources.the childbirth unit has the last generation’s facilities for measurement of Transient Evoked Oto-Acoustic Emissions (TEOAE) only. The test is executed by the paediatrics nurse, paediatrician, and/or the audiometrist, audiologist or ENT specialist, after an appropriate training.the childbirth unit has the last generation’s facilities for measurement of TEOAE and the equipment for automatic auditory brainstem responses (AABR) or clinical auditory brainstem responses (ABR). The test is executed by the audiometrist, the audiologist or, eventually, from the ENT specialist, the paediatrician, the paediatrics nurse, after an appropriate training.the structure has the last generation facilities for TEOAE and AABR in childbirth clinic; further the structure is provided with an audiological ward including the equipment for clinical ABR with threshold definition, clinical TEOAE, Distortion Produced Oto-Acoustic Emissions (DPOAE), impedance audiometry and behavioural audiometry; furthermore the structure has experienced personnel in the early diagnosis of hearing loss, in the rehabilitation of children with HA or CI, and it has a suitable arrangement for the execution of aetiological investigations refer cases; a dedicated structure for collect babies with refer results can be arranged.The screening procedure should be always executed within the discharge of the baby from the childbirth unit, except for few complex situations (e.g. newborn voluntary early discharge). In those situations an appointment for the screening procedures is given at the discharge, according to the audiological centre’s personnel. The childbirth centre repeats the test within 2 weeks from the birth of the babies for verify the refer at NHS. In the 0,42% of all the children underwent the NHS, has been made a final diagnosis of hearing impairment [In the paper published by our group in 2011 the 1,04% of neonates born in the Neonatal Unit of Santa Chiara Hospital in Pisa, underwent a further audiologic assessment after been defined as Each hospital must identify for each childbirth unit a person in charge of screening’s procedures and databases; this professional would be a paediatrician or another specialist , and he/she would involve nurses and/or audiometrists in the execution of the screening’s tests.We suggest that each childbirth centre selects qualified personnel for the execution of the tests . Generic operators has to be avoided.All the childbirth centres with an high number of births per year, the centres with a neonatal intensive care unit (NICU), the II and the III level centres should have a continuous connection with the Audiology/ENT unit and dedicated audiomestrists. Further, specific training courses can be arranged for the operators in each local area.The access at screening’s procedure for children born in private hospitals in Tuscany region has been established, as well as the procedures for children born at home.Furthermore, babies born in other Italian regions or abroad and adopted children that did not attend the screening procedures, must undergo hearing screening (or other audiologic tests adequate for the age of the subject) in the audiologic centre, within one month from the birth or from the taken in charge from the paediatrician.Is it necessary to differentiate the execution’s modality of the UNHS in newborns with or without risk’s factors for auditory neuropathy (AN). In Table pass) on both ears the procedure is concluded. If in one or both the ears no TEOAEs (refer) are measured, another measurement in both ears [pass), the procedure is concluded. If TEOAEs are absent in one or both ears and if personnel and facilities are disposable for the measurement of AABR (level II centre), an AABR measurement is done within the discharge of the baby from the childbirth unit; if AABR are normal (pass) the procedure is concluded. In fact, AABR (with stimulus level set at 45 dB HL) has got an high specificity, providing a very small number of false positive cases. TEAOEs, indeed, is a test more sensible then the AABR, but it can present an higher number of false positive cases. For that reason we decided to consider pass at screening a newborn with pass AABR and refer TEAOEs, but we also considered necessary that those babies has to be closely followed by the paediatrician.TEOAE in childbirth centres are measured by the responsible personnel within the discharge of the baby from the childbirth unit generally after 24 h of life. The TEOAE should be measured during the spontaneous sleep of the baby after the nutrition, possibly in silence. If the TEOAEs are present  within a month from the birth.Cases that still result Babies resulted refer at II level screening’s procedure (TEOAE + AABR) are addressed within III month from the birth, to III level centres for a diagnostic-audiologic complete assessment . Then, further diagnostic sessions, hearing aids fitting and rehabilitation procedures can be performed, and within the 6th month from the birth of the baby a therapeutic and rehabilitative treatment has to be assessed. In newborns with severe-profound bilateral hearing loss, an as-early-as-possible (3rd - 4th month) hearing aids fitting and speech therapy is desirable to reduce as much as possible the time of hearing deprivation.refer at I level screening with TEOAE must be submitted to CMV DNA research in urine by PCR within the 15th day from the birth.All the newborns resulted report UNHS results on baby’s paediatrics bookletreport on the register of childbirth centre the result, the execution’s modalities of the screening and the category of the baby: with or without risk’s factors for auditory neuropathy, with or without risk’s factors for progressive or late onset hearing impairmentreport the screening’s result in an online secured databaserefer UNHS, the childbirth centre’s personnel must inform to the parents, verbally and with an informative paper about the instruction for the II level evaluative testsinform the parents and the paediatrician of hearing screening’s results. Further, if the baby result having previously contacted the responsible audiologic centre, the personnel must inform the parents about the appointment for the II or III level evaluative tests (an informatics booking system between the audiologic centre and the childbirths centre is suggested)the II level evaluative tests have to be executed within a month from the discharge from the childbirth centre and the audiological diagnosis has to be accomplished within 3 months from the discharge from the childbirth centreAt the discharge from the childbirth centre, the person in charge of the screening procedure or its collaborators must:refer at NHS, a new retest in the audiologic centre is scheduled before the discharge. Further the responsible personnel constantly check the database, verifying that all the refer cases execute the audiologic evaluation.The booking system and the database permit to reduce lost to follow-up cases. In fact for each newborn resulted A distinction between the execution of screening protocol in newborns with and without risk’s factors for auditory neuropathy is needed. So, newborns with the characteristics reported in Table All the newborns that show the condition reported in Table If the newborn is preterm, the screening’s procedure has to be executed at the end of the 35th gestational week or after, but within the discharge, in order to reduce the incidence of false positive; before the 35th week, indeed, the execution of TEOAE may be difficult for the limited dimensions of the external auditory canal and the AABR’s responses may be not revealed for an immaturity of the central hearing pathways.refer, is suggested to execute the tests at least 2 times before the discharge. For this procedure is strongly suggested a collaboration with skilled personnel (in particularly with audiometrist).Babies belonging at the categories reported in Table refer at TEOAE must execute, within the 15th day from the birth, the research of CMV DNA by PCR in urine.Newborns resulted refer for one or both ears are addressed to the III level centre for an audiological evaluation.Babies resulted At the discharge, the responsible personnel for the screening procedure or its collaborators must follow the guidelines reported in the previous paragraph at point 1, 2, 3, 4, 5, 6.Babies included in risk categories that was born in I level centre, must undergo AABR in II or III level centre to complete the screening’s procedure within a month from the birth.Children with risk’s factor for progressive or late-onset hearing loss Table  have to The personnel in charge of screening’s protocol have to inform the paediatricians about the inclusion of babies in the group of patients at risk for progressive or late-onset hearing loss, during the discharge of the baby from the childbirth centre.Children that acquire post-natal infections associated with sensorineural hearing loss  or suffer for cranial trauma  or sustain chemotherapy (e.g. with cisplatinum) or treatments with ototossic drugs (e.g. aminoglicosidis), have to undergo an audiological evaluation. Further, an audiologic assessment has to be executed in every cases in which the family or the educators suspect an hearing impairment or in every case of a delay in speech development Table .Table 3RFurther all the newborns admitted to an hospital during the first months of life, in whom occur hyperbilirubinemia that require exanguino-transfusion, sepsis with positive coultural examination, bacterial meningitis, since those conditions are risk’s factors for hearing loss, must undergo a new screening with TEOAE and AABR within the discharge.The paediatrician has a key role in the supervision of the execution of screening’s procedure, in the execution of an audiologic follow-up and in the surveillance of children at risk of progressive or late-onset hearing loss .Moreover in the new protocol for UNHS in Tuscany region is suggested that the paediatrician execute a constant audiological surveillance with specifically developed surveys. These surveys have been developed with the collaboration of the family paediatricians of the Tuscany region and are aimed to identify red flags of hearing loss. Beside having a key role in identifying cases with acquired, progressive or late-onset hearing loss, the family pediatrician has a key role in the identification of false negative cases  and they represent a large proportion of hearing impairment in children (20–30%), even if reliable international data on the subject are not available , 6.In the same way, babies with acquired hearing loss  are not identified by the screening, and those cases can be precociously identified by the audiological surveillance’s programs , 6.The JCIH in 2007 Position Statement, identified the problems related with progressive, late-onset and acquired hearing loss, and it defined the risk’s factors in which is recommended an audiologic follow-up in the first years after the birth . The claFor some of the risk’s factors reported in the 2007 JCIH position statement, the real possibility to cause progressive or late onset hearing loss has not been determined clearly in scientific literature yet, or it is considered not-significant or very weak . In fact, the most recent studies in scientific literature tend to simplify the list of risk’s factors for progressive or late-onset hearing loss; it also facilitate the execution of a more selective and effective follow-up protocol , 6, 8.Moreover, in 2007 JCIH statement, risk’s factors are reported in the same list, those present at birth  and those occurred in the perinatal period which can lead to progressive or late-onset hearing loss (e.g. prematurity associated conditions). Acquired conditions, indeed, should be considered independently, since they require a different surveillance’s protocols.An audiologic evaluation within the 24 and the 30 months for children with low risk’s condition for progressive or late-onset hearing loss, as suggested by the JCIH (2007) may be excessive, difficult and useless, causing an excessive work loading for the Sanitary System with a not clear cost/benefit rate. We believe that in those cases a surveillance by the general paediatrician could be extremely useful, with the submission of specifically developed questionnaires to parents. Babies identified in this surveillance protocol and children with speech delay must be addressed by the paediatrician to the responsible audiologic centre, for the deepening of the evaluation with tests appropriated for the age.Due to these critical issues and after a revision of the recent literature on the subject, we considered to modify and simplify the list of risk’s factors, classifying them into two different distinct tables: risk’s factors for progressive and late-onset hearing loss Table  and acquAnother issue related to UNHS program is represented by the possibility to miss some newborns with light or mild hearing deficits, limited to high frequencies. In fact, tests used for the I and II level screening (TOEAE and AABR), even if very reliable, cannot identify such deficits, for technical limits of the tests.Another possible cause of missed identification of an hearing impairment is related with the auditory neuropathy (AN); in fact a screening executed with the measurement of TEOAE only is not capable to identify the affected cases. There are some cases in which newborns have an higher risk to develop AN. The JCIH statement of 2007 did not specifically enlist the risk’s factors for AN. In the new Tuscany region’s guidelines, such risk’s factors have been identified, taking inspiration from the audiological risk’s factors reported in the Position Statement of JCIH, with some modifications and simplifications Table . NewbornWe believe that for these mentioned reasons a program of audiological follow-up and surveillance is crucial. In Italy each child is addressed to a general paediatrician, so the it plays a key role in the audiological follow-up and surveillance. In Tuscany region’s screening protocol, the role of the general paediatrician is underlined and it is suggested that the paediatrician execute an audiological surveillance during the periodical controls with a specifically developed survey, reported in Additional file Another considerable issue in the audiological newborn screening protocols and in the early identification and treatment of infantile hearing impairments is represented by the congenital CMV infection (cCMV) .Nowadays cCMV infection is considered the first non genetic cause of infantile deafness and it is an important reason for many infantile neurodevelopmental disorders; anyway, since most of newborns with cCMV is symptomless at birth and since a screening protocol for cCMV does not exist yet, the actual impact and the real frequency of this important infection are not yet precisely defined.For a certain diagnosis of cCMV a strict temporal window is available; it is limited at the first 2 or 3 weeks after birth. After this period is difficult to discriminate from a congenital and an acquired infection. Those diagnostic issues, together with the lack of symptoms at birth in most of newborns with cCMV, are responsible of many cases of misdiagnosed congenital CMV infection . So, theFurther, is it well known that is extremely important to identify a cCMV as soon as possible. Indeed, many cases of cCMV related deafness are progressive or late-onset and so, an early identification of cCMV, makes possible the execution of an audiological follow up. Further, an early diagnosis, within the first month from the birth, make possible to execute, in selected cases, an antiviral drug treatment in an optimal temporal window .As far as we know, the NHS protocol of the Tuscany regions is the first to propose and implement a screening for CMV congenital infection in all the newborns refer at TEOAE measurement, by the research of CMV genome by PCR on urine. This procedure permits an early diagnosis of an eventual cCMV; further it allow to avoid false positive results and it resolve the diagnostic doubts on the possibility of a post-natal infection; furthermore it is important for making possible to set up an audiologic follow up in affected babies and for guarantee the chance to submit the affected babies to the recent therapeutic strategies with antiviral drugs.refer at NHS. The study concluded that the screening for cCMV, executed on saliva of babies resulted refer at NHS is feasible and well accepted from the families and, above all, it permits to identify cases that may benefit from antiviral drugs. Recently our group published a study on the incidence of cCMV in preterm and small for gestational age (SGA) babies, hospitalized in Neonatology Operative Unit of Azienda Ospedaliero Universitaria of Pisa (AOUP) that have been submitted at screening protocol. An high incidence of cCMV has been found in preterm  and SGA  babies. The babies resulted positive for cCMV resulted also refer at TOAE measurement in the 25% of cases and at ABR meas in 16,7%. According on this study it seems to exist an important association between cCMV and prematurity or SGA condition at birth [Williams et al.  have recat birth .The execution of UNHS is the only way to identify newborns with congenital moderate to profound hearing loss. Progressive or late onset hearing loss represents a relatively large proportion of childhood hearing impairments and may be not identified by the screening, as well as acquired hearing loss, light-mild hearing loss or impairments limited to the high frequencies and hearing loss related to AN or neural deafness. To early identify those cases an audiological follow-up program is necessary, as well as an audiological surveillance protocol.The new NHS protocol in Tuscany region considered all the mentioned issues, as well as it underscores the role of the general paediatrician in supervising the screening execution and the audiological evaluation, and the audiological follow-up and surveillance.refer at TEOAE measurement are early investigated for CMV congenital infection by the research of CMV DNA in urine by PCR. In Table Finally, in consideration of the importance of cCMV as a cause of hearing loss in childhood, in Tuscany region all the newborns resulted


CCL5 appeared to associate with Müller cells and RGCs as well as synaptic connections between horizontal cells and bipolar cells in the OPL and amacrine cells, bipolar cells and RGCs in the IPL. Although all three high-affinity receptors  for CCL5 were expressed constitutively, CCR5 expression was significantly higher than CCR3, which was also markedly greater than CCR1. Localization patterns for constitutive CCR5, CCR3 and CCR1 expression differed, particularly with respect to expression in inner retinal neurons. Stress-related expression of CCL5 was primarily altered in aged DBA/2 mice with elevated IOP. In contrast, changes in expression and localization of both CCR3 and CCR5 were evident not only in aged DBA/2 mice, but also in age-matched control mice and young DBA/2 mice. These groups do not exhibit elevated IOP, but possess either the aging stress (control mice) or the genetic predisposition to glaucoma (DBA/2 mice). Together, these data indicate that CCL5 and its high-affinity receptors are constitutively expressed in murine retina and differentially induced by stressors associated with glaucomatous optic neuropathy. Localization patterns further indicate that CCL5 signaling may be relevant for modulation of synapses in both health and disease, particularly in the inner plexiform layer.Signaling by inflammatory cytokines and chemokines is associated with neurodegeneration in disease and injury. Here we examined expression of the β-chemokine CCL5 and its receptors in the mouse retina and evaluated its relevance in glaucoma, a common optic neuropathy associated with sensitivity to intraocular pressure (IOP). Using quantitative PCR, fluorescent  Glaucoma is a chronic, age-related optic neuropathy, and the second leading cause of blindness worldwide . IrreverCytokine signaling is a key component of the glial response in glaucoma and several cytokines are implicated as mediators of RGC survival, including Tumor Necrosis Factor alpha ,6 and thChemokines are organized based on the position of cysteine residues, and their biological effects are mediated through seven transmembrane-spanning G-protein-coupled receptors (GPCRs) ,17. VariHere, we sought to characterize expression of CCL5 and its high-affinity GPCRs in murine retina and discern the potential relevance of CCL5 signaling to glaucoma. Using a variety of molecular and histological techniques, we found that CCL5 and its high-affinity receptors are constitutively expressed in the murine retina and are differentially induced by both aging- and IOP-related stressors associated with glaucoma. CCR5 appeared to be the predominant receptor for constitutive CCL5 signaling, but CCR3 may be particularly important for CCL5 signaling in the inner retina. In both constitutive and stressor-induced contexts, localization patterns for signaling machinery indicates likely relevance for CCL5 signaling in synaptic layers of retina, particularly the inner plexiform layer.-/-; 4 month) were obtained from Jackson Laboratories  and bred in-house at the Vanderbilt University animal facilities.Male DBA/2J and age-matched control C57BL/6mice were both obtained at 1 month of age from Charles River Laboratories  and individually housed for the duration of the experiments. CCL5-deficient mice , as previously described ,40. MeanFor analysis of fresh retina tissue, mice were sacrificed by cervical dislocation followed by decapitation. Whole eyes were enucleated, flash-frozen and stored at -80°C until RNA isolation. For tissue procurement for paraffin-embedded and wholemount retina analyses, mice received an overdose of pentobarbital  and were sacrificed by transcardial perfusion with PBS  followed by 4% paraformaldehyde . Whole eyes were enucleated and post-fixed for one hour in PFA. For paraffin retina sections, eyes were paraffin-embedded and 6 μm serial sections of the entire globe were obtained. For wholemount preparations, whole retina was dissected from the eyecups and vitreous removed.Total RNA was isolated from retina with Trizol  and treated with DNase I, as previously described ,9,41. QuTo quantify CCL5 in retinal lysates (5 retina/sample), soluble extracts were isolated using the ProteoExtract Trans-Membrane Protein Extraction Kit  and analyzed using the Milliplex™ Map Mouse Cytokine/Chemokine Magnetic Bead Panel immunoassay kit . Quality controls and standards were run concurrently with experimental samples. Each control, standard and sample was run in duplicate. Total protein concentrations were determined using a BCA Protein assay .3VO4, 0.25 mM PMSF, and protease inhibitor cocktail (Roche). Protein concentration was determined with a Bio-Rad protein assay . Samples (60-80 μg of protein) were prepared in denaturing buffer containing: 100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 200 mM dithiothreitol and separated by SDS-PAGE. Membranes were blocked for one hour using Odyssey TBS Blocking Buffer  followed by primary antibody incubation overnight at 4°C  using the QuantiGene ViewRNA ISH Tissue Kit, according to manufacturer's instructions . Briefly, paraffin sections from C57BL/6and CCL5-/- mice were deparaffinized at 60°C for 1 hour, washed in xylene, and postfixed in 4% PFA. Tissue was incubated with 1× pretreatment solution at 95°C, followed by a protease digestion at 40°C for 10 minutes. Tissue was washed three times in PBS and incubated with alkaline phosphatase-conjugated CCL5 probes at 40°C for 2 h. Following the 2 h incubation, tissue was washed and placed in storage buffer overnight. Fluorescently labeled substrate (Fast Red) was used to detect CCL5 probes followed by immunohistochemistry with primary antibody (see below) against the Müller cell marker glutamine synthetase  a DAPI counterstain, and visualized using Nikon Ti microscope (Nikon Instruments) and analyzed using the NIS Element software program (Nikon Instruments).To assess CCL5 mRNA, we performed a fluorescent Paraffin-embedded Retinal Sections. To evaluate the expression of CCL5 and its receptors, immunohistochemistry (IHC) against CCL5, CCR1, CCR3 or CCR5 was performed on paraffin-embedded, longitudinal sections of retina, as previously described . Brieflyvia the Costes method. Verification and statistical assessment of co-localization was conducted by Fay method randomization, in which pixel co-localization in each channel was compared to those in at least 25 randomized image shifts. P value of 1.00 represents loss of co-localization in the randomized images, which indicates true co-localization of pixels in the original image. P values of less than 0.95 indicate retention of co-localization in randomized images at greater than 95% confidence, indicating the potential for erroneous co-localization of pixels between the two samples. Tresholded Mander's split co-localization coefficients were used to compare co-localization patterns of CCR3 and CCR5 with cell type-specific markers. For Mander's split co-localization coefficients, a value of zero equals no co-localization and a value of one equals complete co-localization. Values reported indicate the proportion of CCR3 or CCR5 labeling that co-localizes with the given cell type-specific marker.Co-localization of labeling between CCR3 or CCR5 and cell type-specific markers was conducted in Image J. Scatterplots of fluorescent intensity across each image were generated with auto threshold determination CCR1, CCR3 and CCR5 were normalized to and compared among groups using either a one-way analysis of variance with pair-wise multiple comparisons by Bonferroni test or one-sample T-test. For immunoblotting, densitometry measurements were compared across groups. Mander's split co-localization coefficients for cell type-specific markers were compared for CCR3 and CCR5 by one-way analysis of variance with pair-wise multiple comparisons by Bonferroni test. For all analyses, p ≤ 0.05 was considered statistically significant.All statistical tests were performed with SigmaPlot . For CCL5 secretion, mean pg/ml/μg total protein values were compared using Student's t-test following normality test validation. For qPCR, mean delta cycles above threshold (Ct) values of We first examined expression and localization of CCL5 in retina from naïve C57Bl/6 mice. Using quantitative PCR, we found that CCL5 is constitutively expressed in healthy murine retina . CCL5 mRα+ cells in the OPL and IPL (To identify cell types closely associated with the CCL5 protein, we co-immunolabeled longitudinal sections of C57Bl/6retina with antibodies against CCL5 and cell type-specific markers for horizontal cells, amacrine cells, rod bipolar cells, RGCs and Müller glia . Confoca and IPL , process and IPL , soma of and IPL  and proc and IPL .To determine potential targets for CCL5 in murine retina, we examined whole retina gene expression as well as layer-specific protein expression of all three high-affinity receptors, CCR5, CCR3 and CCR1. Quantitative PCR in whole retina from naïve C57BL/6mice revealed that all three high-affinity receptors are constitutively expressed in murine retina, with greater expression of CCR5 than both CCR1 and CCR3 p<0.01; . Immunobvia the Costes method. As such, our assessment is limited to qualitative association. We found that CCR1 immunolabeling most associated with calbindin+, GAD65+ and Brn3a+ cells in the GCL (via the Costes method with Fay randomization. In contrast, CCR5 immunolabeling was most strongly associated with GAD65+ processes in the OPL and IPL (via the Costes method with Fay randomization. Despite the appearance of Ccr5 co-localization in the pre-synaptic terminals of PKCα+ bipolar cells, this association achieved a P value of only 0.35 via the Costes method with Fay randomization (To identify specific cell types associated with CCR expression, we performed co-immunolabeling of all three CCRs with cell type-specific markers  for reti the GCL . No stai the GCL . As dete the GCL  and GAD6 the GCL . Weaker  the GCL , Brn3a+  the GCL  and GSyn the GCL . All ass and IPL  and Brn3 and IPL . Weaker  and IPL  and Müll and IPL . These amization . This inmization  are not To quantitatively compare expression profiles of CCR3 and CCR5, we measured the thresholded Mander's split co-localization coefficients of CCR3 and CCR5 with the same cell type-specific markers in serial sections from the same retina. For Mander's split co-localization coefficients, a value of zero equals no co-localization and a value of one equals complete co-localization. In To determine whether glaucoma-related stressors alter expression of CCL5 signaling machinery, we examined protein expression and localization of CCL5, CCR5 and CCR3 in retina from age-matched C57Bl/6and DBA/2 mice. The DBA/2 mouse is an inbred strain that develops elevated IOP with age, resulting from mutations in the GPNMB and Tyrp1 genes ,43. GlauImmunolabeling in whole eye sections were consistent with protein expression studies for CCL5, CCR5 and CCR3. For CCL5, the overall pattern of immunolabeling in young C57Bl/6, aged C57Bl/6and young DBA/2 retina was consistent with those depicted in via the Costes method with Fay randomization. While co-localization was evident in all conditions, the magnitude of CCR3 labeling and the strength of its association with RGCs and Müller cells appeared to differ between conditions. Compared to young C57BL/6 retina, CCR3 co-localization increased with Brn3a, but decreased with GSyn, in aged C57BL/6 retina (Like CCL5, patterns of CCR3 expression also shifted in aging and glaucomatous retina. As determined by co-localization frequencies and illustrated by the intensity of co-localized pixels, CCR3 labeling remained associated with Brn3a+ RGCs and GSyn+ Müller cells in all conditions . These a6 retina . In youn6 retina . In cont6 retina . The ext6 retina . Interes6 retina . In aged6 retina , while C6 retina .via the Costes method with Fay randomization. However, the strength of CCR5 and Brn3a co-localization appeared markedly higher in aged C57Bl/6and young DBA/2 mice (Like CCR3, CCR5 remained associated with Brn3a+ RGCs across all conditions, as determined by co-localization frequencies and illustrated by the intensity of co-localized pixels . This asA/2 mice  than in A/2 mice . Like BrA/2 mice . HoweverA/2 mice . In contA/2 mice . InteresA/2 mice .To quantitatively compare changes in expression profiles of CCR3 and CCR5 associated with glaucoma-related stressors, we measured the thresholded Mander's split co-localization coefficients of CCR3 and CCR5 with Brn3a and GSyn in each condition. For Mander's split co-localization coefficients, a value of zero equals no co-localization and a value of one equals complete co-localization. In Here we examined the potential relevance of the β-chemokine CCL5 in glaucoma. Through a series of expression and localization studies, we found that CCL5 and its high-affinity receptors are constitutively expressed in murine retina and are differentially induced by both aging- and IOP-related stressors associated with glaucomatous optic neuropathy.Constitutively, CCL5 localizes to all layers of the retina, with the potential to influence a variety of neuronal and glial cell types. Based on localization patterns, CCL5 expression appears particularly relevant in the inner retina and synaptic layers. CCL5 associates with Müller cells and RGCs as well as synaptic connections between horizontal cells and bipolar cells in the OPL and amacrine cells, bipolar cells and RGCs in the IPL. Based on expression levels, CCR5 appears to be the predominant receptor for constitutive CCL5 signaling. However, localization studies reveal that CCR3 may be particularly important for mediating CCL5 signaling in the inner retina. CCR1 expression was detectable, but minute, with sparse localization in the inner retina. Although CCR3 and CCR5 are associated with similar cell types in the inner retina, co-localization studies with cell type-specific markers revealed preferential localization of CCR3 to calbindin+ cells and preferential localization of CCR5 to brn3a+ cells and PKCα+ cells. Calbindin is expressed by horizontal cells and some RGCs, while brn3a and PKCα are expressed by RGCs and rod bipolar cells, respectively. Although the functional significance of constitutive CCL5 signaling in retina is not yet clear, constitutive signaling in brain is associated with both neuronal development ,49 and nTo determine whether CCL5, like the β-chemokine Ccl2 ,25, is iIn addition to changes in overall expression, it is likely that the functional consequences of aging- and glaucoma-related CCL5 signaling differ from that of constitutive signaling. This is at least partially supported by dramatic variations in expression and localization of both CCR5 and CCR3 in stressed retina. CCR5 expression was greater in DBA/2 mice than C57Bl/6mice, regardless of age. However, neither aging nor elevated IOP altered CCR5 expression levels. In contrast, CCR3 expression increased nearly 2-fold with normal aging in C57Bl/6 mice, but decreased by approximately 50% with aging + elevated IOP in DBA/2 mice. While protein expression studies in whole retina suggest that CCR5 is not impacted by glaucoma-related stressors, localization studies indicated that glaucoma-related stressors alter cell type-specific expression. In particular, CCR5 expression is greatest in Brn3a+ RGCs in young DBA/2 retina and lowest in GSyn+ Müller glia in aged DBA/2 retina. The localization pattern for CCR5 in aged DBA/2 retina suggests that this decrease in expression by Müller glia is ofset by increased expression in other retinal cell types in the OPL, INL and IPL. Based on our localization studies in naïve retina, this is most likely horizontal, bipolar or amacrine cells. In contrast, CCR3 localization was most impacted by normal aging, which induced increased expression in Brn3a+ RGCs and reduced expression in GSyn+ Müller glia. This suggests that CCL5 signaling preferentially impacts neuronal populations in aging retina.In chronic neurodegenerative diseases of the brain, such as Multiple sclerosis, Alzheimer's disease and HIV-dementia, CCL5 signaling is associated not only with its classical definition as a chemoattractant for monocytes -36, but It is important to note that, in both constitutive and induced capacities, several cell types appear to express CCL5 and at least one CCR, including RGCs, amacrine cells, bipolar cells and Muller glia. This indicates that there are a variety of potential autocrine and paracrine mechanisms for CCL5 signaling within and between these cell types in both healthy and pathological conditions. That stressor-related changed the expression and localization profile of CCL5 and CCR suggests that a shift in the nature and directionality of these autocrine and paracrine signaling pathways may be particularly relevant for pathological roles of CCL5 in RGC degeneration.


This review sought to evaluate recent findings in this field, highlighting how among cathepsins, the inhibition of cathepsin S in particular, could play a significant role in diminishing the effects of CVD, especially for patients with CKD.Cardiovascular disease (CVD) is responsible for the majority of deaths in the developed world. Particularly, in patients with chronic kidney disease (CKD), the imbalance of calcium and phosphate may lead to the acceleration of both vascular and valve inflammation and calcification. One in two patients with CKD are reported as dying from cardiovascular causes due to the resulting acceleration in the development of atherosclerosis plaques. In addition, CKD patients on hemodialysis are prone to aortic valve calcification and often need valve replacement before kidney transplantation. The lysosomal proteases, cathepsins, are composed of 11 cysteine members , as well as serine proteases cathepsin A and G, which cleave peptide bonds with serine as the amino acid, and aspartyl proteases D and E, which use an activated water molecule bound to aspartate to break peptide substrate. Cysteine proteases, also known as thiol proteases, degrade protein  Cathepsins are lysosomal proteases that breakdown peptide bonds linked to specific amino acids. They are known for their key role in protein turnover and significantly contribute to the breakdown of the extracellular matrix (ECM). More importantly, cathepsins play a crucial role in various conditions that involve large biological systems such as autoimmune disease, cardiac repair, cardiomyopathy, heart valve disease, and atherosclerosis.The cathepsin family, cysteinyl, in specific, is known to date to be composed of 11 members in humans including cathepsins B, C, F, H, K, L, O, S, V, W, and Z. Cathepsin S is thought to be a particularly potent cysteine protease cleaving elastin and generating bioactive elastin peptides, leading to the promotion of cardiovascular inflammation and calcification. Beyond boosting inflammation as a part of its own mechanistic process, cathepsin S is also released by smooth muscle cells and macrophages as a systemic response to inflammation in a continuous recursive feedback loop.Vascular inflammation, in particular, is a characteristic feature in the initiation and progression of atherosclerotic lesions. Vascular inflammation that is caused by atherosclerosis is described by widespread changes and remodeling of the ECM within the arterial wall, both in the degradation of the matrix and cellular phenotypic changes. Compounded by chronic inflammation and a consistent imbalance in calcium phosphate serum levels, patients with chronic kidney disease (CKD) face a deleterious feedback loop. This harmful combination further accelerates and exacerbates the evolution of atherosclerosis in patients with CKD. Studies on atherosclerosis-associated inflammatory cytokines have also shown that cathepsin expression and activity are amplified within vascular cells .Also affecting patients with CKD, aortic valve calcification is highlighted as a major clinical problem in this population, where calcification can lead to the progression of aortic valve stenosis. Patients with CKD on hemodialysis are prone to developing aortic valve calcification and likely to need valve replacement before undergoing kidney transplantation. Of further importance is the fact that no current treatment options are available for patients with aortic valve stenosis besides valve replacement. Further, recent clinical trials have shown no benefit to statin therapy in halting the progression of aortic calcification and stenosis , leavingWithin this context, one in two patients with CKD are reported as dying from increased cardiovascular burden particularly resulting from an acceleration in the development of calcification and atherosclerotic plaque . Hence, The inhibition of cathepsin S has been highlighted as a promising intervention in reducing plaque development and diminishing the effects of CVD, especially for patients with CKD . This isA review was conducted through a search of the PubMed, Medline, the Cochrane Library, and Google databases. The following keywords were used: “cathepsin S,” “cardiovascular disease,” “chronic renal disease,” “cathepsin S inhibitor,” “renal disease,” “calcification,” and “atherosclerosis.” The keywords were combined to reach the relevant results.A review of the titles and abstracts was undertaken to find those that matched the keywords of the search. Following the review of titles and abstracts, the authors identified six categories of interest and organized the articles based on this classification: “treatment by cathepsin S inhibitor,” “cathepsin S and renal disease,” “cathepsin S mechanism,” “cathepsin S and calcification,” “cathepsin S and inflammation,” and “miscellaneous.”The initial review of the databases yielded a total of 41 articles. Of those 41, 13 articles were categorized under “cathepsin S mechanism,” 12 under “cathepsin S and inflammation,” 7 articles were categorized under “treatment by cathepsin S inhibitor,” 6 were as “cathepsin S and renal disease,” one under the category “cathepsin S and calcification,” and two under “miscellaneous,”Researchers have sought to understand the role of cathepsin S in CVD and systems by evaluating the molecule’s potential mechanisms of action. One of the systemic mechanisms recently studied is cathepsin S’s role in cardiovascular inflammation and calcification especially for patients faced with CKD.in vivo experiments that elastin degradation induced by cathepsin S accelerates arterial and aortic valve calcification in a CKD model. The findings indicate a mechanism of action where cathepsin S contributes to the cleavage of the elastin matrix in atherosclerotic lesions and inflamed valves, and in the disruption of the tissue layer, mesenchymal cells [vascular smooth muscle cells (SMCs) or valvular myofibroblasts] proliferate and calcify. The researchers highlight that CKD patients with systemic mineral imbalance or hyperphosphatemia experience further acceleration of calcification. The investigators conclude that future interventions in calcification-prone individuals should target inflammation and phosphate imbalance to reduce the effects of negative feedback loops related to elastin degradation and calcification, and incorporate the selective inhibition of cathepsin S as a treatment target.Aikawa et al.  demonstrOn the other hand, in an arterial medial calcification (AMC), a different type of vascular calcification, uremic mouse-model fed a high-phosphate diet, Pai et al.  suggest in vitro. These findings suggest that elastin degradation could in fact induce calcification.Nevertheless, Simionescu et al.  in a stuAdditionally, in a murine model with cathepsin S inhibitor E64d, Chen et al.  find thaSasaki et al.  suggest Cheng et al.  in a comIn abdominal aortic aneurysm (AAA), often described for its wide degradation of the aortic wall matrix, remodeling and further rupturing of the wall, Qin et al.  in a murImportant to highlight is that investigators suggest future interventions in calcification-prone individuals target inflammation and phosphate imbalance, together with the selective inhibition of cathepsin S as a treatment target , 11. AddOther researchers have also inquired about various cathepsin S inhibitors as treatment for cardiovascular disorders. Ahmad and Siddiqi  perform In addition, Rupanagudi et al. , in an eFurther, Payne et al.  assess tHilpert et al.  further The described research has looked at various cathepsin S inhibitors such as RO5444101, RO5461111, LY3000328, among others, to understand mechanism, evaluate clearance from plasma, test toxicity, and investigate whether these inhibitors may be good options as clinical treatment targets. Researchers have also demonstrated how the inhibition of cathepsin S decreased the size of atherosclerotic plaques showing cathepsin S’s potential role as a therapeutic option.In terms of the association between cathepsin S and inflammation, Jobs et al.  investigFurther, in two independent cohorts of elderly men and women, Jobs et al.  sought tLafarge et al. , in a reAdditionally, Naour et al.  in a proAikawa et al.  demonstrFurther, Carlsson et al.  in a lonCardiovascular calcification is described as a disease resulting from the disarray of an individual’s mineral metabolism where the buildup of minerals such as calcium and phosphate can lead to the vessel hardening and disruption of normal physiological processes . At the Further, of important clinical concern has been the progression of calcification to even more debilitating conditions such as plaque rupture or aortic valve stenosis. Of additional concern is the fact that there are currently no treatment options available for reducing or avoiding these advanced conditions, beyond valve replacement .in vitro. These findings suggest that elastin degradation could in fact induce calcification of vascular SMCs and valvular myofibroblasts and thus mediate calcification.In a study looking at the role of fibroblasts in medial vascular calcification, Simionescu et al.  find thaA few other studies have also suggested that in the presence of mineral discrepancy, cathepsin S can participate in cardiovascular calcification where extracellular vesicles could then serve as loci for microcalcifications –49. The Multiple studies have indicated that cathepsin S activity increases with the progression of CKD and have highlighted cathepsin S inhibition as a therapeutic target in the prevention of cardiovascular complications for this patient population. Patients with CKD face a deleterious feedback loop composed and compounded by chronic inflammation and a consistent imbalance in calcium phosphate serum levels. This harmful combination accelerates and exacerbates the evolution of atherosclerosis in patients with CKD. In atherosclerotic lesions, cathepsin S, one of the most potent mammalian elastases, is highly expressed. Macrophages are then set in action as an essential part of the innate immune response, further releasing high levels of cathepsin S.With the progression of kidney failure in patients with CKD, uremia and uremic toxins, produced during the deterioration of multiple biological functions could lead to the apoptosis and early damage of vessel wall. Extracellular vesicles could then serve as loci for microcalcifications –49. HoweCathepsin S, through the cleavage of elastin and generation of bioactive elastin peptides, boosts cardiovascular inflammation and calcification. Changes in mineral imbalance, along with the elevation of cathepsin S, may lead to the transformation of mesenchymal cells, including vascular SMCs and interstitial cells to an osteochondrogenic, or stiffened phenotype, thus accelerating calcification even further Figure  3). Thr. Thr3). via increased elastolytic activity and production of elastic peptides that directly induce smooth muscle cell or interstitial cell differentiation toward osteogenic bone-like phenotype, or via the induced release of extracellular vesicles or apoptotic bodies that could serve as loci resulting in dystrophic calcification (Therefore, as demonstrated in these investigations, cathepsin S may play a deleterious role and be the culprit for the initiation of calcification. This transition to a calcified state could be induced either fication .Aortic valve calcification through the hardening of the valve reduces the movement of aortic valve leaflets, impacting and weakening cardiac function. Because mature aortic valves have an elastin-rich, multilayered structure and can develop inflammatory lesions that recapitulate features of atherosclerotic plaques, researchers have proposed that similar mechanisms of cathepsin S-associated elastin degradation contribute to the development of calcific aortic valve disease .Facing this deleterious feedback loop compounded by chronic inflammation and a consistent imbalance in calcium–phosphate serum levels, patients with CKD are found in a harmful combination that would further accelerate and exacerbate the evolution of cardiovascular calcification , 52. TheThe studies evaluated in this review have provided further evidence of the potential of the inhibition of cathepsin S as intervention toward reducing plaque development and diminishing the effects of CVD especially for patients with CKD.BS: data analysis and manuscript writing; JF: data analysis; EA: critical review of the manuscript, supervision, and funding.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Following current prognosis, demographic development raises expectations of an aging of the working population. Therefore, keeping employees healthy and strengthening their ability to work, becomes more and more important. When employees become older, dealing with age-related impairments of sensory functions, such as hearing impairment, is a central issue. Recent evidence suggests that negative effects that are associated with reduced hearing can have a strong impact at work. Especially under exhausting working situations such as working overtime hours, age and hearing impairment might influence employees’ well-being. Until now, neither the problem of aged workers and long working hours, nor the problem of hearing impairment and prolonged working time has been addressed explicitly. Therefore, a laboratory study was examined to answer the research question: Do age and hearing impairment have an impact on psychophysiological and subjective effects of long working hours. In total, 51 white-collar workers, aged between 24 and 63 years, participated in the laboratory study. The results show no significant effects for age and hearing impairment on the intensity of subjective consequences  of long working hours. However, the psychophysiological response  to long working hours differs significantly between hearing impaired and normal hearing employees. Interestingly, the results suggest that from a psychophysiological point of view long working hours were more demanding for normal hearing employees. Until now, neither the problem of aged workers and long working hours, nor the problem of employees suffering from hearing impairment and prolonged working time has been addressed explicitly in a psychophysiological approach. Moreover, hearing impairment is one of the prominent symptoms of increasing age. The current paper addresses this interplay in a laboratory study.Demographic development raises expectations of an aging of the working population in the next years e.g., , making Apart from the previously discussed impact on the quality of life of the concerned person itself, uncorrected reduced hearing also influences all persons that want to communicate with persons with hearing impairment . One canAccording to article 2 of the Directive 2003/88/EC of the European Parliament and of the Council of 4 November 2003 concerning certain aspects of the organization of working time . An exacPrevious research has shown that the organization of working time has not only an effect on health and social contacts/the social environment, but is also important from an economic point of view. For example, In addition to previously described general effects of age, age related impairments and long working hours, also the interplay with employees’ recovery, is of interest. Following According to As mentioned before, long working hours can provoke stress. On the one hand, stress can be assessed subjectively . On the other hand, psychophysiological parameters, such as cardiovascular activity and responses of the endocrine system are able to show stress or rather arousal of an individual from an objective point of view. Regarding the endocrine system, mental, emotional, and physical stress can lead to an increase in cortisol levels within minutes. This is one of the reasons why the psychophysiological parameter cortisol is applied as one of the most important stress hormones besides the catecholamines noradrenaline and adrenaline . CortisoAeq, for hearing impaired compared to normal hearing participants. The authors assume that this effect is possibly caused by the fact that hearing impaired are being more noise sensitive and distracted by noise than normal hearing individuals. Controversial results for the relationship between (long) working hours and cortisol levels are shown in literature. Whereas effects of gender, smoking status, alcohol consumption or job support were often investigated in the different studies, the combined effects of long working hours, age and hearing impairment were not investigated as important variables – at least not in a psychophysiological approach. If age was mentioned in the studies, the authors report that age was controlled within their samples. Our study extends past research on long working hours by including age as variable of interest. Therefore, the aim of the study presented in this paper was to examine if age and hearing impairment, do have an impact on psychophysiological and subjective effects of long working hours. Accordingly, the following research question should be answered: Do age and hearing impairment have an impact on psychophysiological and subjective effects of long working hours?A laboratory study with repeated measurement design was chosen. The study was conducted in an experimental laboratory of the department of psychology at the University of Graz, Austria.M = 39.69, SD = 11.62). Following the definition of M = 11.47, SD = 7.06). The criterion to be included in the hearing impaired group was a worse ear hearing loss of 15 dB or more on a minimum of two out of the four (speech relevant) frequencies 0.5, 1, 2, and 4 kHz. Following the criterion, 21 participants  were included in the hearing impaired group, 30 participants  were included in the normal hearing group. The unequal distribution regarding age of the two groups, t(49) = -3.38, p = 0.001, can be explained by the natural aging process of auditory functions. To control possible effects of the commute of the participants from their workplace to the experimental laboratory, a baseline measurement of all study variables was performed at the beginning of the experimental session. Regarding the study variables, no significant differences at the baseline measurement were found for the two different hearing groups. Therefore, the effects can be attributed to the study manipulation. In addition the hearing groups do not differ significantly regarding average overtime per week in the last 3 months nor regarding the number of overtime hours in the current working week in which they participated in the study.In total 61 employees (white-collar workers) participated in the laboratory study. Salivary cortisol measurements of 51 of them were available. Loss of salivary cortisol data of 10 participants was due to not analyzable samples or the non-willingness to participate in this psychophysiological measurement. Therefore, for analyses presented within this paper, the final sample consists of 51 white-collar workers, aged between 24 and 63 years  frequencies 0.5, 1, 2, and 4 kHz)] was defined as independent variable and age of the participants was included as covariate in the analyses. Five different dependent variables (DV) were measured: DV1: perceived recovery, DV2: perceived fatigue, DV3: subjective emotional well-being, DV4: subjective physical symptoms, DV5: cortisol level.Time of measurement was used as repeated measurement factor. Overall, each study participant participated in the study for two consecutive days. To participate in the study, each study participant came to our laboratory of the University of Graz directly after his/her regular working hours. On both days the participants had to work in the study  at least three additional hours to simulate a situation of long working hours . The examination procedure is based on the “Grazer fatigue paradigm” , which iTwo measurement times were analyzed for all dependent variables : (1) at the end of the study session of the first day of the laboratory study, and (2) at the end of the study session of the second day of the laboratory study. In addition, a third measurement time was available for saliva cortisol level: the waking cortisol level on the second day of the study.Pure-tone audiometry was conducted in the beginning of the first study session, using a standard Audiometer . Following the WHO  and the To measure cortisol in saliva, saliva samples were obtained with Salivette tubes (Sarstedt) at three measurement times during the study: (1) at the end of the study session of the first day of the laboratory study, (2) the waking cortisol level on the second day of the study and (3) at the end of the study session of the second day of the study. Measurement time (1) and (3) were instructed by the investigator. Only measurement time (2), the waking cortisol level had to be carried out independently by the study participants. The participants were instructed to take the saliva sample 30 min after waking up in the morning and not to brush their teeth, eat, drink, smoke, or do physically demanding activities before providing the saliva sample. The exact time had to be recorded in a protocol. The saliva samples were analyzed at the Technical University of Dresden  by a professional blind to the experimental conditions. There, the samples were centrifuged for 5 min at a rotation speed of 3,000 rotations/min. The cortisol concentration was measured using cortisol luminescence immunoassay (CLIA) with a high sensitivity of 0.16 ng/ml.Feeling of emotional well-being  … 0  to 6 (most intensive).” Two measurement times were analyzed: (1) at the end of the study session of the first day of the study, and (2) at the end of the study session of the second day of the laboratory study.For the measurement of the current subjective emotional well-being a category adjective checklist (German version) consisting of 24 items (BSKE-24-ak) from For the measurement of perceived recovery, the German version of the scale for perceived recovery (SwE) from For the measurement of perceived fatigue, the German version of the scale for perceived fatigue (SwM) from Feeling physical weakness or physical exhaustion … 0  to 6 (most intensive).” Two measurement times were analyzed: (1) at the end of the study session of the first day of the study, and (2) at the end of the study session of the second day of the laboratory study.For the measurement of the current subjective physical symptoms the 24-item German version of the multidimensional physical symptom list (MKSL-24-ak) from The statistical analyses of the data were conducted using the software SPSS for Windows. MANCOVAs and ANCOVAs with repeated measures were used as statistical procedure. The analyses were based on a significance level of 5%.F = 5.42, p = 0.024, Table 1. Regarding the sub-dimension irritability, correlations between the age of the participants and their reported irritability at the end of the study sessions show that irritability tends to decrease with age .The results of a MANCOVA with repeated measures showed a significant effect of the covariate age. Following the univariate tests for the eight sub-dimensions, this effect reaches the level of significance only for the sub-dimension irritability, Table 1.Hearing impairment does not have a significant effect neither on the perceived recovery of the participants nor on their perceived fatigue at the end of the study sessions. Also, the covariate age does not significantly impact the results and all within-subject effects did not reach the 5%-level of significance. The coefficients are shown in Table 1).The result of a MANCOVA with repeated measures showed no significant effect of hearing impairment for subjective physical symptoms. Also, the covariate age as well as effects of measurement time did not reach the 5%-level of significance (see Table 1). The descriptive statistics are shown in Table 2. Hearing impaired employees tend to show lower cortisol levels than normal hearing employees. The effect is most pronounced for the waking cortisol level of the second day (t2). Follow-up analyses (ANCOVAs) that were conducted for each time point, show that the effect only reaches the level of significance for this time of measurement (see Table 3). Hearing impaired employees show a significant lower waking cortisol level than normal hearing employees.The results of an ANCOVA with repeated measures showed a significant effect for the covariate age and for hearing impairment. Furthermore, the effect time x hearing impairment just failed significance. The results of the analysis of covariance indicated that although age had a significant effect on the cortisol level, group differences remained significant. All other effects do not reach the 5%-level of significance , no significant group differences can be shown in our study. Furthermore, with one exception for the sub-dimension irritability of the subjective emotional well-being, the covariate age does not have a significant impact on the subjective results. Regarding the sub-dimension irritability it can be shown that irritability tends to decrease with age. However, the findings of the current study do not support the previous research results that older employees seem to have a stronger need of recovery than younger employees e.g., . FurtherInterestingly, data of salivary cortisol reveal that the non-significant subjective results are not supported by the objective physiological saliva cortisol data. A significant effect of hearing impairment was shown for the cortisol level. Furthermore, the effect time x hearing impairment just failed significance and a significant effect for the covariate age can be reported. The results of the analyses indicated that although the covariate age had a significant effect on the cortisol level, group differences remained significant: Hearing impaired employees tend to show lower cortisol levels than normal hearing employees. The effect is most pronounced for the waking cortisol level of the second day (t2). Follow-up analyses that were conducted for each time point showed that the effect only reaches the level of significance for this time of measurement. Hearing impaired employees show a significant lower waking cortisol level than normal hearing employees. Following Furthermore, the idea arises that following This study has some limitations. First, our study participants had to work long working hours on two consecutive days. Especially regarding the non-significant subjective effects but also regarding effects on the cortisol level see also , it is sSecond, we did not test our participants on “normal” working days without working overtime hours. We therefore recommend that future research shall include days without extended working hours as additional control condition. Other possibilities to enable a comparison between normal and long working hours are the inclusion of a baseline measurement of cortisol level before the sessions of additional work and/or the inclusion of a control group working normal working hours, into the study design.One limitation of the study is that young/old comparisons in psychophysiological parameters can always be questioned, as the physiological system and the systems reactivity normally change with increasing age .Furthermore, from a practical point of view it would be interesting to examine possible demanding working situations in future studies that are more and more typical for a global working world, like audio-conferences during unusual working hours  and/or in foreign languages. Another facet with practical implications is the impact of environmental conditions of the workplace like noise or lack of space or privacy like it could be found in open-plan offices. In addition, the investigation of different categories of work  should be addressed in future studies.The results of our study show no significant effects for age and hearing impairment on the intensity of subjective consequences of long working hours. But, age and hearing impairment do matter from a psychophysiological point of view. Psychophysiological responses  on long working hours differ significantly between hearing impaired and normal hearing employees. Interestingly, the results suggest that long working hours were more demanding for normal hearing than for hearing impaired employees. Furthermore, normal hearing employees tend to show a higher waking cortisol level after 1 day with long working hours than hearing impaired employees. To uncover possible long-term effects further research is still required.This study was carried out in accordance with the recommendations of the ethics committee of the University of Graz with written informed consent from all subjects. The study was approved by the ethics committee of the University of Graz.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


R)-2-methoxyethyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate hydrochloride (ET-26 HCl) is a novel etomidate analogue. The purpose of this study was to characterize whether ET-26 HCl could retain the superior myocardial performance of etomidate in vivo and in vitro. channel.In vivo, after a single bolus administration of ET-26 HCl or etomidate, no significant difference in echocardiography and electrocardiogram parameters was observed. No arrhythmia occurred and no QT interval prolongation happened during the study period. In the in vitro Langendorff preparation, none of the cardiac parameters were abnormal, and the hERG recordings showed that ET-26 HCl and etomidate inhibited the tail current of the hERG in a concentration-dependent manner with an IC50 of 742.51 μM and 263.60 μM, respectively.in vivo experiment and a whole organ preparation, the current study found that ET-26 HCl can maintain a myocardial performance that is similar to that of etomidate. In addition, the electrophysiology study indicated that ET-26 HCl and etomidate inhibited the hERG at a supra-therapeutic concentration.In conclusion, through an  Etomidate [R-1-(1-ethylphenyl) imidazole-5-ethyl ester]  is an imR)-2-methoxyethyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate hydrochloride (ET-26 HCl)  have developed a series of etomidate analogues, from methoxycarbonyl-etomidate, carboetomidate, and methoxycarbonyl-carboetomidate to cyclopropyl-methoxycarbonyl metomidate, which are designed not to suppress adrenocortical function –10.Our l-26 HCl)  has beenKr), encoded by the human-ether-a-go-go-related gene (hERG), which plays a critical role in defining ventricular repolarization .–24.in vi3 25.0, KCl 4.7, KH2PO4 1.2, CaCl2 2.5, MgSO4 1.2, glucose 11.0, EDTA-Na2 0.125 and the pH was maintained at 7.40 ± 0.05. The solution was continuously oxygenated using a mixture of oxygen (95%) and carbon dioxide (5%) in a 37°C water bath .A Langendorff heart preparation  was used to evaluate the effects of ET-26 HCl and etomidate on cardiac performance. A total of 36  isolated rat hearts were randomly assigned to an ET-26 HCl group  or etomidate group . After anesthetising the rats using sodium pentobarbital , the rat hearts were quickly removed and mounted on a Langendorff perfusion system via the aorta at a constant pressure of 70 mmHg using Krebs-Henseleit buffer containing (in mM): NaCl 118.0, NaHCOmax), were measured before treatment (baseline) and at 1, 3, 5, and 10 min after hypnotic treatment. The experimental protocol is shown in After a 30-min stabilization period, ET-26 HCl or etomidate was administered into the inflow perfusate at a concentration of 5, 10 or 30 μM. Cardiac parameters, as follows: (1) heart rate (HR); (2) left ventricular systolic pressure (LVSP), and (3) maximal rate for left ventricular pressure rise and decline  cells were elicited by a 2 s depolarizing pulse to +50 mV from a holding potential of −80 mV and repolarization to −50 mV for 3 s to measure the tail currents in an interval of 30 s. In the absence and presence of ET-26 HCl and etomidate, their effect on the hERG tail current was recorded. Based on a preliminary experiment, the used concentrations were 30, 100, 300, 1000, and 3000 μM for ET-26 HCl and 10, 30, 100, 300, and 1000 μM for etomidate. The inhibition percentage (%) of the hERG tail current was calculated using the following equation: inhibition percentage (%) = {1 − (remaining current amplitude)/ (control current amplitude)} × 100. The results are presented as mean ± standard deviation (SD). The ICin vivo are presented as mean ± SD. The differences among groups were compared using independent-samples t-tests. Repeated-measures ANOVA was performed to analyse the changes in several variables over time, such as LVEF, LV internal dimension and end-diastolic/systolic volume. For the in vitro Langendorff preparation, data are presented as mean ± SD relative to baseline values (percentage) and analysed using independent-samples t-tests. P < 0.05 is considered statistically significant. The statistical analysis was performed using the Statistical Package for Social Sciences (SPSS™), Windows version 16.0 .Data concerning the cardiac parameters in vivo hemodynamic study, a total of 18 Beagle dogs, with a mean weight of 9 kg (range 7–10.5 kg) and age of 8–10 months, were used to evaluate the influence of ET-26 HCl and etomidate on cardiac function using echocardiography and ECG. For the 50 of ET-26 HCl and etomidate, showing that the echocardiographic parameters at 1, 3, 5, and 10 min after hypnotic administration are similar to the pre-dose recording. After treatment using different doses of ET-26 HCl and etomidate, no significant change was found in TTE parameters . Fig 3 din vitro cardiac function  was found between the two groups  and age of 8–10 weeks, were used. Before treatment, the basal cardiac parameters were recorded. After treatment using ET-26 HCl or etomidate at 5, 10, or 30 μM, no significant change in o groups .i.e., etomidate at concentrations of 10, 30, 100, 300, and 1000 μM reduced the tail current by 3.83 ± 2.36%, 14.13 ± 5.09%, 27.03 ± 4.24%, 51.72 ± 4.12% and 80.06 ± 3.26% (n = 3), respectively. The IC50 value was 263.60 μM.50 value was 742.51 μM.The etomidate analogue, ET-26 HCl, inhibited the hERG current in a similar concentration-dependent manner . ET-26 Hin vivo and in vitro. The major finding of the present study was that ET-26 HCl retains the superior myocardial performance of etomidate. To the best of our knowledge, this is the first investigation to define the effect of this etomidate analogue on cardiac performance using echocardiography, combined with assessments of ventricular repolarization and proarrhythmic risk, and an electrophysiology study, to provide new experimental evidence for cardiac safety.The purpose of this study was to evaluate the effect of a single administration of ET-26 HCl on cardiac function when compared with etomidate 50, the in vivo hemodynamics study demonstrated no significant differences in echocardiography and ECG parameters. LV internal diameter, LV volume in diastole and systole and LVEF remained in the normal range.After a single bolus administration of 1×, 2×, 4× EDin vivo study, no arrhythmia occurred, and no prolongation of the QT interval was found.It is still unclear whether prolongation of the QT interval is associated with the occurrence of drug-induced TdP –27, but in vitro hemodynamics study was also designed to compare the direct cardiac effects of ET-26 HCl and etomidate by analysing responses at equimolar concentrations. Cardiac function was assessed by measuring LVSP and ±dp/dtmax. The results demonstrated that etomidate, and its analogue, ET-26 HCl had similar effects on the isolated rat hearts perfused at constant pressure.The in vitro concentrations of etomidate were chosen to correspond with peak plasma concentrations reported in vivo in humans. The recommended intravenous dose for induction of anesthesia is approximately 0.3 mg/kg for etomidate, and the peak plasma concentration (Cmax) during induction has been reported to be approximately 3 μM, and researchers have shown that the concentration of etomidate which causes sinus arrest in all hearts is 1000 μM [in vitro rat hemodynamics study, and 10–3000 μM for the in vitro electrophysiology study.The  1000 μM –29. Therin vitro IKr assay and the whole-cell patch-clamp technique to study the effects of ET-26 HCl and etomidate on the hERG channel. The results suggest that etomidate and ET-26 HCl inhibited the tail current of the hERG in a concentration-dependent manner, but the concentrations decreased the channel protein expression at a supra-therapeutic concentration or overdose, with an IC50 of 263.60 μM and 742.51 μM, respectively.Correspondingly, we designed the 50 value in vitro may be misleading, as multiple factors, such as hypokalaemia, structural heart disease or other cardiac ion channels can mitigate or worsen the risk of QT prolongation [50 measurements must be interpreted in the context of Cmax. If the safety margin (safety margin = IC50/Cmax) is 30 or greater, the incidence of TdP is low [While a general relationship between hERG blockade and proarrhythmic risk is generally accepted, an isolated ICongation –31. TherP is low –33. Althin vitro Ikr assay, we did not further elucidate cellular mechanisms affecting repolarization and compound activity at other cardiac ion channels. Therefore, future investigations should proceed on the basis of the present study.There are also several potential limitations in the current study. Firstly, in order to rule out the possible influence of drug interactions, isolated rat hearts and dogs were selected at random for each sub-group, and each rat or dog was used only for one concentration or dose of one drug, and not for rising concentrations or dosages at intervals of several minutes; therefore, the curves might not reflect a good dose-dependent relationship. Secondly, in the in vivo experiment and a whole organ preparation, we found that ET-26 HCl can retain the superior myocardial performance of etomidate. In addition, the electrophysiology study indicated that ET-26 HCl and etomidate inhibited the hERG at a supra-therapeutic concentration.In conclusion, using an S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S10 Table(PDF)Click here for additional data file.S11 Table(PDF)Click here for additional data file.S12 Table(PDF)Click here for additional data file.