Sirtuins are a class of histone deacetylases (HDACs) that have been shown to regulate a range of pathophysiological processes such as cellular aging, inflammation, metabolism, and cell proliferation. There are seven mammalian Sirtuins (SIRT1-7) that play important roles in stress response, aging, and neurodegenerative diseases. However, the location and function of Sirtuins in neurons are not well defined. This study assessed the retinal expression of Sirtuins in mice, rats, and humans and measured the expression of Sirtuins in aged and injured retinas. Expression of all 7 Sirtuins was confirmed by Western blot and Real-Time PCR analysis in all three species. SIRT1 is highly expressed in mouse, rat, and human retinas, whereas SIRT2-7 expression was relatively lower in human retinas. Immunofluorescence was also used to examine the expression and localization of Sirtuins in rat retinal neurons. Importantly, we demonstrate a marked reduction of SIRT1 expression in aged retinal neurons as well as retinas injured by acute ischemia-reperfusion. On the other hand, none of the other Sirtuins exhibit any significant age-related changes in expression except for SIRT5, which was significantly higher in the retinas of adults compared to both young and aged rats. Our work presents the first composite analysis of Sirtuins in the retinal neurons of mice, rats, and humans, and suggests that increasing the expression and activity of SIRT1 may be beneficial for the treatment of glaucoma and other age-related eye dysfunction. The Sirtuins (SIRT) are an NAD+ dependent class III HDACs that share extensive homology with the yeast HDAC Silent Information Regulator 2 (Sir2) . SIRT/SiThe Sirtuins are involved in cell and tissue metabolism, making the study of Sirtuin expression in the retina important given that the retina is one of highest energy-consuming tissues in the body . SIRT1 hSirtuins are expressed throughout the brain , ears, muscle, liver, kidney, vasculature, skin, lungs, and other locations in the body . SIRT1 iC57BL/6 male mice, 6–8 weeks of age; and Sprague-Dawley (SD) male rats, 2–3 months of age were obtained from Center of Laboratory Animal Science of Nanchang University. For aging study, the rats were used in the following age groups: young (2 months of age), adult (12 months of age), and old (20 months of age), which are roughly equivalent to human aged 20, 40, and 80 year old, respectively . All proSix pairs of adult donor eyes (age range 30–50 years) were obtained from Red Cross Society of China Jiangxi Branch. All experiments involving human samples were approved by the Ethics Committee of Affiliated Eye Hospital of Nanchang University. All volunteer signed informed consent documents in written form in accordance with the principles of the Declaration of Helsinki. To obtain fresh eye tissues for RNA/protein and staining, we restricted the globes in the present study to those received less than 48 h postmortem. The previous medical and ocular histories of all donors were assessed to exclude donors with any eye disease. The retinas were processed as described below.Sprague-Dawley rats, 2–3 months of age were used. Under anesthesia , transient, unilateral, retinal ischemia was produced in rat eyes. Briefly, the anterior chambers of both eyes were cannulated and the IOP in one eye was elevated above systolic blood pressure (∼110 mm Hg) for 60 min and the contralateral eye was cannulated and maintained at normal IOP . Rats weTable 1) were synthesized by TINGEN Biotech, Beijing, China. Real-time PCR was performed using the RealMasterMix (SYBR Green) kit  with the following conditions: denature, 95°C, 20 s; annealing, 58°C, 20 s; and extension, 68°C, 30 s. All samples were tested in triplicate PCR reactions and the mean of the reactions was used for calculating the expression levels. All the data were collected from the linear range of each amplification. Expression levels were normalized to the average of tubulin mRNA levels from the same samples.Total RNA was extracted from each mouse, rat, and human retina using TRIZOL (Invitrogen) according to the manufacturer’s instructions. The concentration of isolated RNA was quantitated spectrophotometrically. Two hundred nanograms of total RNA from each sample was reverse transcribed into cDNA using the Quantscript RT Kit . Specific primers for each gene (Table 2) followed by incubation with peroxidase-conjugated secondary antibodies . Finally, proteins on membranes were detected with enhanced chemiluminescence western blotting detection reagents (EMD Millipore).Retinal tissues were dissected from the retinal pigment epithelium and choroids and lysed in the RIPA buffer containing PMSF . The lysates were centrifuged, and supernatants were collected and subjected to western blot analysis. Protein concentrations were measured using the Bradford assay (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Sample proteins (30 μg) were separated by 7.5–15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The blots were blocked in 5% non-fat dry milk in Tris-buffered saline/Tween-20 and incubated with the appropriate primary antibodies  at a sub-boiling temperature for 10 min followed by cooling 30 min. Then the tissue sections were incubated with specific primary antibodies (Table 2) diluted in 5% bovine serum albumin (BSA) in PBS for overnight at 4°C. The sections incubated with PBS without primary antibody were used as negative controls. After several wash steps, the tissue sections were incubated with the secondary antibody, Alexa Fluor 594 donkey anti-rabbit IgG (H+L) for 1 h at room temperature. The sections were counterstained with DAPI  and coverslipped. The staining was repeated three or more times for each antibody and the results were consistent.For immunohistochemical analysis, the animals were deeply anesthetized with chloral hydrate and then perfusion fixed through the left cardiac ventricle with ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate saline buffer . After perfusion, eyes were enucleated and the anterior segments were removed. The posterior segments were fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer saline for 2 h. After washing in PBS, the tissues were transferred to 70% ethanol overnight, then dehydrated and embedded in paraffin. The 5-μm-thick paraffin tissue sections were dewaxed and then rehydrated. Endogenous peroxidase activity was blocked by placing the sections in 3% Ht-test or Chi square test as appropriate. P < 0.05 was considered significant.All quantified data represented an average of at least three samples. SPSS 16.0 and Graph Pad Prism 5.0 software (Image J) were used for statistical analysis. All data are expressed as mean ± SD. Significance was established by paired Figure 1). The Sirtuins’ mRNA levels were different in each species. The mRNA level of SIRT1 was the highest in rat and human retinas, while SIRT2 was the highest in mouse retina. The mRNA level of SIRT7 was the lowest in rat and human retinas. SIRT3 mRNA was lowest in mouse retina. The mRNA levels of SIRT5, SIRT7, SIRT4, SIRT1, and SIRT6 were found in descending order in the mouse retina. Our results show that SIRT2, SIRT5, and SIRT7 were expressed primarily in mouse retina. Differences in the mRNA levels of SIRT2, SIRT3, and SIRT5 were not obvious in rat retina, while SIRT1, SIRT4, and SIRT6 were expressed at higher levels. The mRNA level of SIRT2 was almost as low as SIRT7 in human retina, while the mRNA levels of SIRT3, SIRT6, SIRT5, and SIRT4 descended in that order. We also discovered that SIRT1, SIRT3, and SIRT6 were primarily expressed in mouse retina . Importantly, we confirmed that all seven Sirtuins were expressed in the retinas of all three species, and the high level of Sirtuin mRNA expression in the retina is consistent with the retina being one of the highest energy-consuming tissues in the body . Sirt3 protein was detected in mouse retina, and was observed at a low level in rat retina . Expression of Sirt4 and Sirt5 in human retina was not obvious whereas their expression was more visible in mouse retina than in rat . Sirt6 and Sirt7 were expressed in mouse, rat, and human retina, and were lowest in human retina . Our results demonstrate that the protein levels of the Sirtuins in mouse retinas descend in the following order: SIRT3, SIRT1, SIRT5, SIRT4, SIRT7, SIRT2, and SIRT6 . The protein levels of the Sirtuins in rat retinas descend in the following order: SIRT4, SIRT1, SIRT2, SIRT5, SIRT7, SIRT6, and SIRT3. Finally, the protein levels of the Sirtuins in human retinas descend in the following order: SIRT3, SIRT4, SIRT1, SIRT2, SIRT7, SIRT5, and SIRT6 . Remarkably, we found the protein levels of the Sirtuins, including that of SIRT1 and SIRT3, but especially SIRT5 and SIRT6, in human retina did not correspond to their respective mRNA levels .The protein expression levels of Sirt1 and Sirt2 were highest in the retina of rats, while their levels were lowest in human retina . SIRT1 expressed in the GCL layer, inner nuclear layer, outer nuclear layer and retinal pigment epithelium. SIRT2 mainly expressed in the GCL layer, outer plexiform layer and retinal pigment epithelium. Meanwhile, SIRT3 mainly expressed in the inner plexiform layer, outer plexiform layer and retinal pigment epithelium, but was less expressed in the GCL layer. SIRT4 mainly expressed in the GCL layer and inner nuclear layer in the form of threadiness. SIRT5, SIRT6 and SIRT7 mainly expressed in the GCL layer, inner plexiform layer and retinal pigment epithelium. From the overall analysis, all Sirtuins except SIRT3 were expressed in the GCL layer, while all Sirtuins except Sirt4 were expressed in the retinal pigment epithelium.Since all Sirtuins were expressed in rat retina, we selected normal adult rat retina for immunofluorescence detection. We observed that SIRT1, SIRT 6, SIRT7 were localized in the nucleus, SIRT2 was predominantly in the cytoplasm, SIRT3, SIRT4, SIRT5 were localized in the mitochondria . SIRT1 protein levels also decreased in the retinas following acute retinal ischemic injury . On the other hand, SIRT3-5  were all expressed at the highest level in adults but were not significantly lower in old rats , while the expression levels of SIRT2, SIRT6, and SIRT7 did not significantly change with age .Prior studies have suggested that SIRT1 is involved in retinal neuron degeneration during aging  and retiTo date, the significance of Sirtuin expression in the retina is unknown. To establish a basis for studying the pleiotropic functions of the Sirtuins in the retina, we analyzed the expression pattern and location of the Sirtuins (SIRT1-7) in the aging retinas of different species at the mRNA and protein level. Our results demonstrated that all members of the Sirtuin family are detected in the retinal neurons of mouse, rat, and human, which is in agreement with previous observations that, in the mice retina, certain cells express SIRT1 . MoreoveIn the present study, we demonstrated that certain Sirtuins are abundantly expressed in the retinas of mouse, rat, and human, and there are some similarities in the expression patterns of Sirtuins among different species. However, there is also considerable variability in the localization and distribution between Sirtuin family members. We found that the mRNA and protein content of SIRT1, which is the earliest discovered and most studied member of the Sirtuin family, is abundant in mouse, rat and human retinas. In previous studies, SIRT1  and SIRTInterestingly, SIRT3 protein was not only detected in mouse, rat, and human retinas, but was highly expressed in human retinas with different antibodies. Overexpression of SIRT3 increases mitochondrial respiration and reduces reactive oxygen species (ROS) production, which may explain its high expression in human retina in order to protect the organ from oxidative stress-induced injury . NotableIn the retinas of mice, rats, and humans, SIRT1 can be observed in the GCL, INL, ONL and RPE, whereas other studies have reported that SIRT1 is only expressed in the GCL, INL, and RPE of rat retinas  or in thWe found that SIRT3 is mainly in the GCL, IPL, OPL, and RPE of rat retinas. Previous reports have demonstrated that SIRT3 is present in the mitochondrial matrix and can regulate mitochondrial function . FurtherS-nitrosocysteine, and therefore PTMs could also be responsible for the two SIRT1 bands seen in young adults. It is interesting to speculate why the lower SIRT1 band is only present in young rat retinas, which also have the highest overall SIRT1 protein levels, as it may represent a more active form of SIRT1 that provides additional protection to the developing retinal neurons.Previous studies have shown that SIRT1 plays a crucial role in age-related retinal degeneration . Its actInterestingly, although SIRT1 decreased with age while SIRT2, SIRT6, and SIRT7 did not change significantly during aging in the rat retina, the mitochondrial Sirtuins  were expressed maximally in adult retinas. This might be due to an increased demand for the antioxidant activities of the mitochondrial Sirtuins during adulthood in the retina, perhaps because of an enhanced reliance on mitochondrial oxidative phosphorylation at this life stage. The subsequent drop in expression of the mitochondrial Sirtuins in old rat retinas could also be partly responsible for age-related retinal dysfunction, and future studies should look to increase their expression in old rat retinas along with SIRT1 to determine which will have the most beneficial effects for delaying retinal neuronal functional decline.To date, calorie restriction (CR) is the most well-known and accepted effective means to extend the lifespan without genetic or pharmacological intervention. The expression of all Sirtuins except SIRT4 increase as an effect of CR . SIRT6 hOur study has provided foundational knowledge about Sirtuins in the retinal neurons of humans, mice, and rats, which will be helpful for future investigations on retinal metabolism and Sirtuin regulation. The present study also provides information on specific Sirtuin family members that may be targeted pharmacologically to combat diseases of retinal neurons. Future studies on Sirtuins should examine their precise protective roles in retinal neurodegeneration and the mechanisms by which they mediate such protection to inform development of treatments for age-related retinal decline.The study was conceived and designed by XZ and HL. Data collection and analyses were conducted by HL, MZ, KJ, JZ, WD, SF, TS, and XZ. The paper was written and revised by HL and XZ.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. The reviewer SK and handling Editor declared their shared affiliation.


Vestibular schwannomas (VS) expand slowly in the internal auditory canal, in the cerebellopontine angle, inside the cochlear and the labyrinth. Larger tumors can displace and compress the brainstem. With an annual incidence of 1:100,000 vestibular schwannoma represent 6–7% of all intracranial tumors. In the cerebellopontine angle they are by far the most neoplasm with 90% of all lesions located in this region. Magnetic resonance imaging (MRI), audiometry, and vestibular diagnostics are the mainstays of the clinical workup for patients harboring tumors. The first part of this paper delivers an overview of tumor stages, the most common grading scales for facial nerve function and hearing as well as a short introduction to the examination of vestibular function. th annual meeting of the German Society of Oto-Rhino-Laryngology, Head and Neck surgery a special volume of the journal “HNO” will be printed. Hence this presentation has been designed to deviate from the traditional standard which commonly consists of a pure literature review. The current paper was conceptually woven around a series of interdisciplinary cases that outline examples for every stage of the disease that show characteristic results for management options to date. Systematic clinical decision pathways have been deduced from our experience and from results reported in the literature. These pathways are graphically outlined after the case presentations. Upholding or improving quality of life is the central concern in counseling and treating a patient with vestibular schwannoma. Preservation of neuronal function is essential and the management options – watchful waiting, microsurgery and stereotactic radiation – should be custom-tailored to the individual situation of the patient. Continuing interdisciplinary exchange is important to monitor treatment quality and to improve treatment results. Recently, several articles and reviews have been published on the topic of vestibular schwannoma. On the occasion of the 88Important criteria for decision making are size and growth rate of the tumor, hearing of the patient and the probability of total tumor resection with preservation of hearing and facial nerve function, age and comorbidity of the patient, best possible control of vertigo and tinnitus and last but not least the patient’s preference and choice. In addition to this, the experience and the results of a given center with each treatment modality will figure in the decision making process. We will discuss findings that are reported in the literature regarding facial nerve function, hearing, vertigo, tinnitus, and headache and reflect on recent studies on their influence on the patient’s quality of life. Vertigo plays an essential role in this framework since it is an independent predictor of quality of life and a patient’s dependence on social welfare. Pathognomonic bilateral vestibular schwannomas that occur in patients suffering from neurofibromatosis typ-2 (NF2) differ from spontaneous unilateral tumors in their biologic behavior. Treatment of neurofibromatosis type-2 patients requires a multidisciplinary team, especially because of the multitude of separate intracranial and spinal lesions.Off-label chemotherapy with Bevacizumab can stabilize tumor size of vestibular schwannomas and even improve hearing over longer periods of time. Hearing rehabilitation in NF2 patients can be achieved with cochlear and auditory brainstem implants. It. It2]. Ih decade . Regardih decade . Other ah decade , 13], , , . The higHNO that reflects the state-of-the-art based on the relevant international literature. Regarding the management of the disease, numerous articles have been published, among others there is a recent review article in the German journal Hence, we decided to focus in this article less on theory but mostly on practice and interdisciplinarity. In the past, many dogmas overshadowed the treatment of patients with vestibular schwannomas. In the times of microsurgery, for a long time the complete tumor resection was considered as most important to avoid tumor recurrences, sometimes even at the expense of functional preservation. The introduction of stereotactic irradiation and the associated possibility to achieve growth interruption of the tumor for a high percentage with at the same time a low rate of side effects, further increases the claims to microsurgical treatment. Today, the preservation of the neuronal function is most relevant. However, it must not be forgotten that this aim can often be achieved with complete tumor resection. For this reason, it is often not possible to develop ex ante the ideal treatment concept for an individual patient. Recurrences occur after surgery and also after irradiation; even bi-modal, combined treatments are not always successful.Even if a patient had a treatment outcome with a low risk of recurrences and good hearing, persisting tinnitus and vertigo might be remaining disorders that are difficult to tolerate. The management of the single patient will always require an individual concept that will have to be adjusted during the further course Figure 1 .This complexity makes it so difficult to create standardized treatment pathways for this disease. So it seems more important to establish a continuous interdisciplinary dialogue than to establish standards. Being open for treatment concepts that go beyond the control or removal of the tumor and that are oriented at the patient’s quality of life, as well as scientifically elaborating new diagnostic and therapeutic options will determine the future.This present article was conceived around a case collection, in which the reader will find examples for every stage of the disease describing the current microsurgical and radio-therapeutic options of disease management. After the case series, suggestions are found for a classification regarding a reasonable and suitable management of the disease in the sense of decision cascades and clinical treatment pathways. Furthermore, factors are discussed that significantly influence the quality of life of patients suffering from vestibular schwannomas. There will also be an overview about the management of those tumors in patients with neurofibromatosis type 2. Tumor size and expansion play a crucial role in terms of the staging. Several authors have correlated these two criteria with different surgical approaches and treatment indications and outcomes . As an eAs an example for a systematic classification of the hearing function, the guideline of the American Academy of Otolaryngology – Head and Neck Surgery (AAO-HNO) is mentioned . It stagClass A Class B Class C Class D According to this classification, functional hearing is only present in the classes A and B.Regarding data collection in the speech discrimination test, standardization would be desirable, but currently it cannot be achieved. Ideally, a validated word list of at least 50 words in the patient’s mother language should be used, which is offered at a standardized level of 40 dB above the hearing threshold or maximally tolerable loudness . In prosFor the diagnosis as well as post-therapeutic follow-up of patients with vestibular schwannoma, vestibular diagnostics play a key role. In the context of pre-therapeutic examinations, the ENT-specialist is highly involved not only in the evaluation of the cochlear function and the function of the facial nerve, he is also responsible to find the exact diagnosis or the exclusion of a certain disease in cases of vertigo symptoms.Even if nearly all patients report about the main symptom of unilateral hearing loss and tinnitus, about 10–15% of the patients suffer from initially unclear vertigo without hearing loss or tinnitus so that nearly all patients seek advice – sometimes by making some detours – of an otolaryngologist.The low number of patients who initially complain about vertigo as single symptom and who are then diagnosed of vestibular schwannoma in the further course, may be explained by the slow growth of schwannomas and hereby sufficient time for central compensation.If vertigo is observed, patients often describe unsystematic uncertain gait with possible increase in the dark or drift tendency to one side; frequently, they only mention it when they are specifically asked. An exact anamnesis of possible vertigo complaints requires sufficient time and attention as every vestibular test does, i.e. also for patients with vestibular schwannoma.In particular after surgical therapy of vestibular schwannoma, patients report about acute vertigo attacks and postural instability. The intensity and severity of the postoperative vertigo is directly proportional to the preoperative vestibular function that has to be determined before surgery by means of extensive vestibular testing.After clinical examination with Frenzel goggles, those examinations encompass also video-nystagmography at rest or after provocation by head shake, orientation and position test under video-nystagmography, caloric test, video head impulse test, examination of the vestibular evoked myogenic potentials as well as an examination in the rotating pendulum chair and an extensive functional testing, for example by means of sensory organization test.Such a complete test battery requires time and staff and a good cooperation by the patient. If those preconditions are not fulfilled or available, the video head impulse test with testing all semicircular canals together with the VEMP testing of sacculus and utriculus already provides a good and time-efficient overview of the function of the peripheral vestibular organ performed by an experienced examiner and allows possible conclusions regarding the already damaged part of the vestibular nerve. In this way, the exact condition of the actual peripheral vestibular function can be measured and a possibly existing central affection in cases of large vestibular schwannomas as well as an already existing compensation can be revealed. Thus a good estimation of the functional postural situation is obtained.Especially patients with bilateral vestibular problems can be identified who have to expect a severe and complicated postoperative course of the disease. In those cases, special preoperative information and counseling have to take place. If necessary, surgery of a vestibular schwannoma has to be postponed or it seems to be more appropriate that the patient undergoes radiotherapy. On the other hand, it is useful that patients with single-sided preoperative vertigo concerning the side of the schwannoma rather undergo surgical therapy because after transection of the vestibular nerve in the context of resection a better central compensation may probably be achieved. So extensive vestibular diagnostics also play a crucial role for making the best therapeutic decision. But also the possibility to inform patients about the expected postoperative complaints gains in importance with regard to current medico-legal aspects.In general, the vestibular rehabilitation should be introduced very early in the postoperative phase and the patients are invited to undertake as many activities as possible that occupy the vestibular system. It is recommended to provide specific exercise instructions teaching the patients how to include balance exercises as often as possible in their daily life and to perform those exercises also with closed eyes to exclude the merely visual compensation. Also professional physiotherapy and exercise instructions as well as training by means of sensory neuro-feedback methods seem to be beneficial to achieve a rapid central compensation of postoperative vertigo.In this way, the re-integration into daily life can be achieved rapidly and in general the patients are free of complaints after 6–9 months.Beside the regular follow-up of the cochlear function, hearing rehabilitation, and imaging, also a short standardized checkup of the vestibular function should be performed in order to document a course of central compensation in the years after therapy. In cases of deterioration, possibly developing recurrence etc., vestibular diseases may be identified earlier.In summary, preoperatively, the high value of sufficient vestibular diagnostics allows better information and counseling about different therapeutic options and expected side effects; and after therapy, the central objective to provide a quality of life as high as possibly can be achieved.In the context of larger tumors, the function of the facial nerve is often impaired, which has an influence on the treatment approach. To assess the degree of neural paresis, the classification according to a proposal of William House and Derald Brackmann from 1985 could be internationally established Table 1 .Regarding further pre- and intraoperative diagnostic procedures  extensive literature on this topic has been published and will not be further discussed here. The transtemporal approach via the middle fossa is suitable for intrameatal and low-grade extrameatal tumors up to a size of 20 mm and patients with residual hearing . The patThe translabyrinthine approach is suitable for patients with tumors that have already destroyed the hearing ability. Beside dissection of the internal auditory meatus, mastoidectomy as well as labyrinthectomy are performed. The advantages are the secure exposition of the facial nerve and the overview of the posterior fossa. The patient is lying in supine position and the head is turned to the contralateral side. After retroauricular, C-shaped incision and mastoidectomy exposing the sigmoid sinus, the dura of the posterior fossa as well as the facial nerve in its bony canal in the area of the second knee, and the labyrinthine bloc, first the lateral semicircular canal is drilled out, then the posterior and finally the superior semicircular canals follow. Afterwards, the vestibulum can be opened and the internal auditory meatus is exposed. The exposition and opening of the dura are performed until the intervention reaches the cerebellopontine angle , 26]..26].The retrosigmoid approach is especially suitable for tumors with larger extrameatal parts. It allows a secure and wide exposition of the posterior fossa and thus tumor resection with hearing preservation . The facAfter opening the arachnoid mater in the area of the cerebellomedullaris cistern and draining off liquor, the cerebellopontine angle is exposed and the internal auditory meatus can be opened.Instead of repeating findings that are published in the literature in a dry and manual-like manner, this contribution wants to convince mainly with a clear description of a case series and give ideas and hints for optimal information and treatment of affected patients.According to the title of this article, those are exclusively cases in which at least the course of the disease justified treatment. Of course, especially in the context of older patients, most centers will first observe the spontaneous course by regularly performing clinical and imaging examinations. The annual – non-volumetric – growth rates of the tumors reported under strict observation varied between 0.3 and 4.8 mm , 33], , . InteresIf the indication for treatment is made in patients that were initially observed, it occurs in 75% of the cases within the first 5 years .In order not to extend the volume of this article, spontaneous courses will not be included in the case series. Some spontaneous courses of the disease, however, will be described based on tumors that were treated later. Furthermore, a differentiation was made between primarily surgical and primarily radiotherapeutic cases in order to demonstrate as clearly as possible the possible effects of the treatment modalities. The examples of this case series are systematically classified according to the functional hearing abilities of the patients and previous treatment (surgery or irradiation). Functional hearing abilityCase #1, male, age: 52 yearsHistory: Vertigo independent from movement and tinnitus as permanent, high frequency whistling sound on the left side for several monthsHearing unchangedIntracanalicular tumor confirmed by MRI, slightly grown after 6 monthsClinical findings:Paresis of the buccal branch of the facial nerve on the left side (House-Brackmann grade II), no other deficitsHearing: class ACaloric reflex test: side difference of 31% on the left sideMRI:Merely intracanalicular left-sided vestibular schwannoma Figure 4 Intervention:Complete tumor resection by microsurgery with preservation of the BAEP, the inferior vestibular nerve, and the facial nerve, performed via a retrosigmoid  approach.Course:Postoperatively, increased vertigo for 3 weeks, especially in the context of lateral eye movementsFacial nerve similar to preoperative findingsClass A (according to AAO-HNO classification)Tinnitus slightly regressive, in particular at night3 years after surgery, no recurrencesFunctional hearing abilityCase #2, male, age: 33 yearsHistory: First presentation with a history of right-sided progredient vestibular schwannoma known for 4 yearsClinical findings:Recurrent vertigoVestibular test: well compensated, chronic-peripheral vestibular disorder of the right sideHearing: Class A, no tinnitus MRI:Intra- and minimally extrameatal vestibular schwannoma located near the fundus, measuring 9x5x5 mm Figure 5 Intervention:Transtemporal resection with monitoring of the facial nerve and intraoperative BERA on September 16, 2010:CourseNo postoperative vertigoFacial nerve: House-Brackmann grade II; normalization within 8 weeksClass A; unchanged compared to surgeryNo tinnitus5 years after surgery, no recurrences Figure 6  No functional hearing abilityCase #3, male, age: 47 yearsHistory: In May 2013, the patient woke up in the morning with an acute vestibulo-cochlear disorder on the right side. First, severe rotational vertigo with subjective deafness and significant tinnitus on the right side. During therapy with cortisone, the tinnitus improved and the vertigo complaints were regressive. The attempt to perform external round window covering on the right side did not lead to improvement of the deafness.Clinical findings:Massive cochlea-vestibular disorder with labyrinth failure and right-sided deafness. Inconspicuous status of the vestibular nerve Figure 7  Intervention:Translabyrinthine resection of the vestibular schwannoma on the right side with monitoring of the facial nerve and insertion of a Contour Advanced dummy into the cochlea on January 28, 2014.Course:Postoperative status of facial nerve: House grade I, slight vertigo with discrete spontaneous nystagmus to the leftIn the Dyna-CT scan dated January 29, 2014, the dummy was correctly located in the cochleaIn the further course, rapid compensation of the vertigo with constant status of the facial nerveMRI on May 23, 2014 without suspicion for residual tumor or recurrence. Performance of a positive promontory testNo functional hearing abilityCase #4, female, age: 36 yearsHistory: Hearing impairment for several monthsPhone calls are possible using the right earIntermittent vertigoSignificant tumor growth over 6 monthsClinical findings:Romberg’s and Unterberger tests are inconspicuousAudiometric high frequency hearing loss in both ears in the range of 2 kHz, on the right side to –60 dB, on the left side to –35 dBSpeech discrimination of 70% on the right side at 60 dBMRI:Inhomogeneous, intra-/extrameatal tumor on the right side that has not yet reached the fundus (stage T2). CISS images clearly demonstrate a thin layer of CSF between the tumor and the brainstem Figure 8 Intervention:Functional hearing preservation with complete microsurgical resection via a retrosigmoid  access. Functional preservation of the facial nerve (House-Brackmann grade 1)Course:Increased vertigo for 3 weeks after surgery, especially in the context of lateral eye movementsSlightly regressive tinnitus, in particular no impairment at nightAs preoperatively, the facial nerve is inconspicuousClass A (according to AAO-HNO)3 years after surgery, no recurrenceNo functional hearing abilityCase #5, female, age: 55 yearsHistory: First presentation with known functional deafness and recurrent rotational vertigo since several months.MRI of the cerebellopontine angle performed in another institution confirmed the suspected vestibular schwannoma of the right side. Clinical findings:Occasional rotational vertigo and high-grade hearing loss in the right ear with inconspicuous status of the facial nerveVestibular diagnostics: chronic peripheral vestibular disorder of the right side without complete central compensationHearing: Class D Figure 9 Intervention:Translabyrinthine access for resection of the vestibular schwannoma on the right side with insertion of a Contour-Advanced dummy into the cochlea on November 17, 2015Course:Initial postoperative status of the facial nerve according to House-Brackmann grade VI that improved with application of a corticosteroid . On the first postoperative day, already House-Beckmann grade II was achieved. Normalization of the function of the facial nerve until the first outpatient follow-up appointment on December 2, 2015.Hearing: right-sided deafnessVestibular function: increasing central compensation observed in the follow-up examinations on February 26, 2016, and June 6, 2016.Functional hearing abilityCase #6, female, age: 35 yearsHistory: Lumbar puncture in the context of neuro-borreliosis diagnostics.Afterwards vertigo was observed, 2 days later sudden hearing loss and tinnitus that persisted longer so that MRI of the skull was performed.In the further course, numbness of the left side of the face was described, mainly in the innervation area of the N. V/2-3Clinical findings:Whispering was well heard in the left ear.Audiometry revealed class A hearing ability.Tendency to turn to the left in the Unterberger test.Stable Romberg’s test.Hypoesthesia in N. V/2-3 on the left.MRI:Vestibular schwannoma of stage T3 on the left side. The tumor grows to the fundus of the internal auditory meatus. Its cranial part touches the trigeminal nerve at the brainstem on the left  access.Course:Initially, balance problems when walking fast or in the dark.Temporary neck pains and frontal headache, especially under stress.In cases of stress, pains in the area of both earsLeft-sided hearing: sometimes hyeracusis, then phone calls with use of the left ear are uncomfortableAudiometry: –10 dB in the speech field in both ears.Speech discrimination: nearly identical in both earsFacial nerve: House-Brackmann grade INo impairment in daily life and in the jobNo recurrence over 3 years after surgeryFunctional hearing abilityCase #7, male, age: 52 yearsHistory: After left-sided sudden hearing loss, a vestibular schwannoma was diagnosed.In the context of follow-up examinations, the patient complained only about unimportant tinnitus on the left side for 5 years.When the tumor had significantly grown, he decided for surgery.Clinical findings:Hypoacusis in the frequencies above 2 kHz on the left to –50 dB, speech field was nearly not touched.Clinical balance tests were inconspicuous.MRI:Course of a primary intra-/extracanalicular vestibular schwannoma under observation until stage T3 was reached  with only slow recovery due to infusions and dexamethasone. Persisting vertigo and tinnitus.Romberg’s test with instability and tendency to fall until 12 months after surgery, then improvement was observed.The patient was more impaired by the right-sided hearing loss, especially in noisy environment.Apart from that, she felt well, no paresis of the facial nerve.6 months after surgery, BiCROS device was applied, of which the patient was not satisfied.Because of unclear electrophysiological findings and negative promontory test, she decided against cochlear implantation.Finally, she was relevantly more satisfied after implantation of a bone anchored hearing prosthesis (BAHA).Functional hearing abilityCase #9, female, age: 41 yearsHistory: 10 years prior to first presentation, already sudden hearing loss on the right with complete recovery due to infusion therapy.Because of new right-sided sudden hearing loss and whistling tinnitus, MRI diagnostics and identification of a vestibular schwannoma on the right side.Nearly no vertigo (dance teacher!)Clinical findings:Nystagmus to the right when looking to the left.Instability in standing and walking tests.In the Unterberg test, tendency to rotate to the right.Subjectively low-grade hypoacusis on the right.Vestibular test: caloric hypoexcitability on the right side.MRI:Vestibular schwannoma on the right with brainstem compression without hydrocephalus  access. Intraoperative improvement of the acoustically evoked brainstem potentials.Course:Postoperatively, first only vertigo was observed.8 days after surgery, rhinoliquorrhea was found, persisting after removal of a lumbar drainage.14 days postoperatively: suboccipital re-craniotomy and sealing of a fistula at the right-sided internal auditory meatus.Then still headache and facial pain for 2 days, especially around the right eye, then rapid improvement.Immediately after surgery, House-Brackmann grade II of the facial nerve, after 3 months grade I.Vertigo had completely disappeared after 1 year.Hearing ability class A as preoperatively.No tumor recurrence 8 years after surgery.Functional hearing abilityCase #10, male, age: 49 yearsHistory: Increasing hearing loss for 6–7 years on the left side, in the course also pressure sensation in the ear and tinnitus (whistling sound).No vertigo.Audiometry: functional hearing loss (left side), class D.MRI:Large vestibular schwannoma with growth into the fundus of the internal auditory meatus and significant brainstem compression  as well as blood deposits at the tentorium and the falx. Clinically, no cerebellar symptoms.No vertigo.Undisturbed balance.Clinical paresis of the facial nerve, House-Brackmann grade II on the left, after 3 months, grade I.Tinnitus had disappeared.No recurrences after 2 years.Functional hearing abilityCase #11, female, age: 34 yearsHistory: 6 months before first presentation, bilateral hearing loss after common cold.Recovery of the right side to normal level; on the left side, persisting higher-grade hearing loss.No relevant vertigo.Clinical findings:Instable Romberg’s test.Unterberg test with rotation to the right.Left-sided hypoacusis No other deficits.Bilateral papilledema .Directly before surgery nausea and vomiting.MRI:Stage T4b vestibular schwannoma on the left side with compression of the aqueduct and obstructive hydrocephalus.Meningioma in the left olfactory area (not depicted) as additional finding .Recurrence after surgery and radiosurgery, hydrocephalusCase #12, female, age: 65 yearsHistory: Ear noise on the left 1 year before first presentation, improved with pharmacotherapy, then again increasing.Diagnostics by means of MRI after observation of increasing numbness of the whole left side of the face. Further increasing painful dysesthesia in this area.Left-sided hearing loss, phone calls using the left ear were no longer possible.Arterial hypertonia.MRI:Vestibular schwannoma on the left side, stage T3 . At rest, recurrent paresis of the buccal branch of the left facial nerve were observed. 2 months later, again increasing gait instability  and dizziness, in particular with massive episodes when the patient got up from a lying position. She had difficulties to walk at all because of the vertigo. Less headaches, only in the morning and evening.MRI of the skull did not show an increased ventricular width, but a clear growth of the residual tumor in the left cerebellopontine angle. Stop of the use of carbamazepine and gabapentin, then slight improvement of the vertigo symptoms.Meanwhile functional anacusis on the left side and increased paresis of the buccal branch of the facial nerve (House-Brackmann grade II).3. Intervention:Lateral suboccipital re-craniectomy on the left side and subtotal microsurgical tumor resection with preservation of the affected facial, glossopharyngeal, abducens, and trigeminal nerves. Between the meatus and nearly in the center of the cerebellopontine cistern, a very thin tumor layer has to remain on the facial nerve because further resection would not have allowed functional preservation of the nerve.Histology:Schwannomas with significant regressive changes, bleedings of different times as well as slight overexpression of p53 and p21.No anaplasia, no malignancy.Further course:Postoperative paresis of the facial nerve unchanged with grade 2 on the left side. Still slight walking and standing instability and slight vertigo.In the further course, again increasing painful dysesthesia in the supply area of the left trigeminal nerve (VAS 7-8/10 for some hours per day). Recurrent reddening of the left eye. After administration of Lyrica 75 mg and Amitriptyline 50 mg 0-0-1, improvement of the painful dysesthesia.MRI performed 6 months after surgery, did not reveal tumor recurrence.Functional hearing lossCase #13, male, age: 43 yearsHistory: Tinnitus rapidly progressing over 3 months and increasing hearing loss in the right ear so that finally the right ear could no longer be used for phone calls.In addition, progressive gait instability.Clinical findings:Romberg’s test was insecure with tendency to fall. Unterberg test could not be performed because of tendency to fall.High-grade hearing loss on the right.Audiometric inner ear hearing loss, on average >–60 dB in the speech field, right-sided.MRI:Large cystic tumor filling the cerebellopontine angle with brainstem compression and advancing into the right internal auditory meatus .Massively increasing vertigo and in particular image fusion disorder due to gaze-evoked nystagmus to the left. It was caused by a circulatory disorder in the area of the inferior cerebellar peduncle and the cerebellar hemisphere. Additional complication because of gout attack in the right ankle.6 months after surgery only slight tinnitus perceived as weak noise.Improvement of the gait instability and still disturbed image fusion when looking straight forward as well as diplopia and blurred vision when looking to the right without visible weakness of the abducens nerve.Rotatory nystagmus when looking to the right. Slightly delayed diadochokinesia on the right side. No longer relevant vertigo. No longer paresis of the facial nerve (House-Brackmann grade I).Romberg’s test still unstable, but compensated.2 years after surgery, the patient is again full-time working. Romberg’s test is stable, Unterberger test is insecure without tendency to fall and without rotating tendency. MRI did not reveal growth of the residual tumor for 8 years.The term of “radiosurgery” was used by the Swedish neurosurgeon Leksell for a method developed by him in the 1950ies. It was a method of high-precision convergence irradiation with a high dose, that is applied once. The patient's head first had to be fixed in a stereotaxy frame (about 0.3 mm precision) similar to stereotactic surgeries. Since the 1980ies, the stereotactic positioning of the patient is applied with a special, less precise mask fixation (±1-2 mm precision) than SRT for the fractioning that is used in radiotherapy. From 2000 to 2012, we performed RS and SRT with the dedicated linear accelerator Novalis (BrainLAB) - because of the longer follow-ups, the case examples were mostly recruited from the population treated with the Novalis. Since October 2012, we work with the robot-assisted radiosurgery system CyberKnife (Accuray) that allows the radiosurgical precision of about 0.3 mm due to permanent image control even without frame with mask positioning. Instead of SRT, the treatment of larger tumors can be limited to 3 single irradiations, the so-called multisession radiosurgery (msRS).First some examples of mainly typical courses will be presented (cases 1–8), then particular, atypical courses will be described that also include severe single cases (cases 9–14).Functional hearing abilityCase #1, female, age: 41 yearsHistory: 3 years before initial presentation, left-sided sudden hearing loss without remaining hearing impairment. Tinnitus in the left ear as noise, not disturbing. In the course, sometimes vertigo and imbalance. In the MRI follow-up of 09/2012 and 04/2013, significant tumor growth of max. 4 mm to 8x6 mm into the left meatus.Clinical findings:Unterberger test instable with undirected tendency to fall.The audiogram revealed inner ear hearing loss of 55 dB on average in the speech field.MRI:Figure 20 Intervention:Radiosurgery .Course:6 months after the intervention, the hearing ability was unchanged. MRI revealed the typical reaction with central contrast agent reduction and relevant contrast enhancement in the edges showing a transiently rather small tumor. In the further course until 12/2015, regression of the reactive changes and regredient tumor size with still well-being and good hearing ability over meanwhile 2.5 years . Tinnitus and occasional vertigo.Alternating hypo- and dysesthesia of the right side of the face.Clinical findings:Anacusis in the right ear.Labyrinth failure on the right.Unterberger test compensated.nd trigeminal ramus (right-sided).Hypo- and dysesthesia in the supply area of the 2Paresis of the facial nerve (House-Brackmann grade III (right side).MRI:Intra-/extrameatal tumor recurrence (16x9 mm), progressing compared to the previous examination one year before.Intervention:Radiosurgery .Course:7 months after intervention, the patient reported about an improvement of the hypo- and dysesthesia of the right side of the face, the other symptoms were unchanged.In the further course, gradual improvement of the paresis of the facial nerve and significant tumor regression confirmed by MRI .Intervention:Radiosurgery .Course:6 months after intervention slightly increasing hearing loss in the right ear. Disturbed balance did not increase.In the further course, increasing right-sided hearing loss with steep decrease to 70 dB.The vestibular nerve can be stimulated on both sides.Clear tumor regression in the MRI follow-up.4 years after radiosurgery, nearly stable MRI findings and stable clinical course .Course:4 months after intervention, clinically and ENT-specifically no deterioration.In the MRI, typical central reduction of contrast enhancement.In the further course, regression of the reactive changes and regressive tumor size in the MRI follow-up until 3 years after radiosurgery, then stable condition.Slightly progressive hearing loss in the deep and middle frequencies of 50–90 dB, constant over >8 years .Rotational vertigo and balance disorder.Hypo- and dysesthesia of the left side of the face.MRI follow-up showing progressive VS on the left, now with brainstem contact.Intervention:Radiosurgery .Course:6 months after intervention, unchanged symptoms.Hearing ability after >7 years on the left side with pancochlear inner ear hearing loss to 85 dB at 6 kHz, in the speech field about 20 dB poorer than initial audiometry.Clinically, further dysesthesia in the area of the left corner of the mouth and the tongue .Course:Due to vertigo and painful dysesthesia on the right side of the face, already 3 months after intervention the first control MRI was performed. It revealed an extraordinarily severe reaction with central reduction of contrast agent and increased volume.In the further course, nearly complete regression of the complaints.9 years after the intervention, still good hearing ability in both ears , progressive in the further course.Intervention:The patient refused the surgery recommended by the interdisciplinary team.Fractionated stereotactic radiotherapy (SRT) with 27x2 Gy  at a tumor volume of 6.5 cm³.Course:6 months after irradiation, the patient reported about unchanged compensated gait insecurity and occasional dysesthesia on the right side of the face that disappeared in the further course.The MRI control examinations first showed a central hypointensity, then increasing tumor regression to 1.8 cm³ tumor volume.Last follow-up examination in 02/2014 revealed unchanged findings (9 years after SRT)  with 27x2 Gy .Course:7 months after irradiation, the patient reported about an increased hypoesthesia on the right side of the face. Labyrinth failure and anacusis on the right side were unchanged.The first 2 MRI control examinations in 2003 showed the typical, central hypointensity of the contrast agent.In the further course, relevant tumor shrinking to 1.1 cm³ in 09/2011 with clinically stable findings  with 27x2 Gy .Course:In the course, first increasing tumor volume was observed from 3.8 cm³ to 7.3 cm³ after 2.5 years. The recommended surgery was refused; stable clinical findings and constant hearing loss.In the further course, regression of the tumor.Clinically improved hearing loss (–40 dB in the speech field) and occasional balance disorders.The patient is fully able to work .Course:In the course, first increasing tumor volume from 0.6 cm³ to 1.5 cm³ after nearly 4 years; the recommended surgery was refused. Stable clinical findings and constant hearing loss.In the further course, tumor regression with unchanged clinical findings.The patient is fully able to work .Course:First typical course with central hypointensity of the contrast agent after 6 months and then marginal regression until 3 years after intervention.1.5 years later, only an intrameatal residual tumor was found with unchanged clinical findings .Course:After 5 months, the tumor volume appears larger with focal spots of absent contrast enhancement. In the next follow-up examination cystic involutions becomes evident, but the tumor size remains constant.Clinically stable findings.One year later, significant regression to a volume of 0.5 cm³.Subjectively neither vertigo nor balance disorders.Less important tinnitus .Intervention:Radiosurgery .Course:Stable clinical findings with hearing aids on the left side.After 3.5 years, recurrent tumor growth, clinically associated with functional hearing loss and increasing balance disorders. The suggested surgery was refused, the second irradiation was performed with a margin dose of 12 Gy (80%).The clinically painful dysesthesia occurred and convulsions on the left side of the face required pharmacotherapy and local botox injections  with 30x1.8=54 Gy .Course:After 7 months, frequent pain attacks of the right trigeminal nerve, other neurological findings were unchanged.MRI showed a severe reaction on SRT with increasing growth and surrounding edema at that time. In the further course, first regression of the reactive changes with clinical improvement of the balance disorders and no more pain attacks  was measured now at 65% according to Röser (1980)  and a reIn the further course, balance tests did not reveal any hint for peripheral vestibular disorders.Over the time of the regular controls, we offered all available therapeutic options. Because of the missing tumor growth and the moderate clinical complaints that could be compensated by hearing aids regarding the hearing ability, the patient finally decided again for regular tumor observation in the sense of wait-and-scan.Over 5 years, no significant tumor growth was observed without therapy  up to a maximum size of 13x0.4x0.7 mm as well as slight progression of the left-sided hearing loss.No functional deficit of the vestibular nerve, although the balance tests revealed discrete signs of peripheral vestibular disorder on the left side.The patient decided for further observation of the findings. Annually performed examinations showed constant growth of the tumor over 3.5 years (last examination performed in March 2016).Audiology revealed a slight increasing hearing loss up to now 95 dB at 4 kHz with constantly good balance function  will ceCystic vestibular schwannomas may be one of the most demanding challenges of microsurgical tumor resection, also because the affected nerves can be highly split up and run adherently at the cell walls so that also larger vessels are difficult to be separated from tumors .The following decision-finding diagrams  . In a prThe weighting of the single factors influencing the quality of life of patients varies enormously. Vertigo plays a key role as well as an increasing hearing loss associated with social restrictions . In thisIn a meta-analysis including more than 25,000 patients, Sughrue et al. found a reported rate of preserved facial nerves of 85% after middle fossa approach, 81% after translabyrinthine approach, and 78% after retrosigmoid approach . Since tLooking at more homogeneous cross-over studies with all tumor stages, generally in more than 90% of the cases preservation of the facial nerve is anatomically and functionally possible in the context of complete tumor resection , 46], , . At thisAlready at the time of diagnosis, many patients do no longer dispose of functional hearing on the tumor side. In a recent study with partly observed , partly microsurgically treated , or with gamma knife  treated patients with a tumor smaller than 3 cm in diameter had a functional hearing ability on the affected side in about half of the cases  . Eight yEspecially in cases of small tumors, however, an experienced surgeon has a good chance to completely resect the tumor and at the same time to permanently preserve the hearing ability in the affected ear . For tumEven very large tumors (>4 cm in maximum diameter) may be operated in single cases with preservation of the hearing ability . Hence, Vertigo is a symptom that may strongly influence the patient’s quality of life. Only in the last years, it has been placed into the focus of attention . ActuallIn 2014, in the context of a cooperative cross-sectional investigation , 538 patients with unilateral vestibular schwannoma were asked regarding the symptom of vertigo  . The surThe incidence of tinnitus is higher in patients who still have hearing abilities in the affected ear . The occIn the same study of 171 patients with preoperative tinnitus, the cochlear nerve was resected in 85 cases (49.7%) and in 86 (50.03%) it was preserved. Between both groups, there was no significant difference with regard to the presence of postoperative tinnitus. A clear pathophysiological explanation of the phenomenon of tinnitus in patients with vestibular schwannoma has not yet been found. The least clarified up to now is the role of headache and its causes. In a mixed series  of 538 patients, Carlson et al. (2015) found headaches of variable incidence and severity in about 50% 8 years after first diagnosis. In comparison to the control group, the patients with vestibular schwannoma complained twice as much about headaches. The treatment modality did not have a significant influence on these percentages, neither had the choice of the surgical approach regarding the long-term risk of severe headaches. Younger patients, patients with higher scores on the anxiety and depression scales, and pre-existing headache diagnoses were the relevant predictors of long-term headaches, the tumor size had not significant influence . It is wDecision-finding regarding the management of patients with vestibular schwannomas should consider the benign nature of the tumor with mostly slow growth and it should be based on the long-term preservation of the quality of life. Radical tumor resection is certainly justified when a realistic long-term benefit may be expected for the individual patient that outweighs the possible negative effect on the quality of life.On the other hand, the stress of the patient caused by the tumor itself must not be underestimated and the chances of treatment must not be wasted by an unrealistic overemphasis of the risksApart from single cases, it should be the disease itself and not the decision for the treatment or the treatment modality that determines crucially the patient’s quality of life , 42]. A. A42]. ACompetent management of patients with the complex disease of NF2, requires a multidisciplinary team including human genetics, neurosurgery, otolaryngology, ophthalmology, neurology, pediatrics, radiology, pathology, radiotherapy, audiology, psycho-oncology, physiotherapy, and other disciplines . The priIntracranially, vestibular schwannomas and meningiomas show similar average growth rates between 0.30 and 2.57 cm³ per year , 77], , , 79], m, m79], mThe indication for treatment and definition of the extent of the surgical resection follows more complex criteria compared to spontaneous vestibular schwannomas. Relevant factors hereby are the bilateral hearing function, tumor size, tumor extent in direction of the brainstem and the internal auditory meatus, growth rate, the patient’s preferences, and comorbidities. The benefit-risk ratio is clearly defined as the elimination of the functional threat vs. the risk of functional loss by the intervention. Even if surgery of the tumors in NF2 patients is sometimes difficult and complicated, radiotherapy is often no real alternative. Already during 5-years intervals, the tumor control rates (= growth stop and/or shrinking) vary significantly after radiation, probably also independent from the applied radiation dose, between 66 and 100%. Also the hearing preservation rate in those intervals amounts to variable 33–57% , 83], , , 85], , , 87], , , 89], , . A growtRelevant constellations regarding the management in cases of bilateral vestibular schwannomas are displayed in Figure 42 As long as a conservative management is reasonably possible, it will generally be preferred in the sense of long-term functional preservation unless there is the microsurgical possibility to remove a small, strategically well located tumor in a bilaterally hearing patient with hearing preservation , 98], , .In cases of brainstem compressing vestibular schwannomas on both sides and bilaterally functional hearing, in general first a unilateral hearing preserving tumor reduction may be conceived (Figure 42A While in cases of unilateral or bilateral small vestibular schwannomas, generally wait-and-scan strategy is recommended, it might be useful and also possible in exceptional cases to microsurgically explore such a small tumor and to remove it with hearing preservation (Figure 42D In cases of large tumors and still functional hearing in one ear and a small tumor with deafness on the contralateral side, it is reasonable to wait as long as the hearing ability remains stable. If the inner ear hearing loss increases in such a case of a growing large tumor, a tumor reduction with hearing preservation, the removal of the posterior wall of the internal auditory meatus, or a therapeutic attempt with Bevacizumab are valid options.If surgery with functional preservation of the facial nerve is not successful in a patient with neurofibromatosis type 2, an immediate transplantation in the same session allows for the best chances for regeneration. A side-to-end connection between the hypoglossal and the facial nerves within the first year after paralysis of the facial nerve can still lead to an equally good result regarding rehabilitation of the face , 101], , 101], , 103], , 103], . For lonThe authors declare that they have no competing interests.


During a study on high mortality cases of goose embryo in Shandong Province, China (2014–2015), we isolated an H9N2 avian influenza virus (AIV) strain , which was supposedly the causative agent for goose embryo death. Sequence analysis revealed that DP01 shared 99.9% homology in the HA gene with a classic immune suppression strain SD06. To study the potential vertical transmission ability of the DP01 strain in breeder goose, a total of 105 Taizhou breeder geese, which were 360 days old, were equally divided into five groups  for experimental infection. H9N2 AIV (DP01) was used for inoculating through intravenous (group A), intranasal instillation (group B), and throat inoculation (group C) routes, respectively. The geese in group D were inoculated with phosphate buffer solution (PBS) and those in group E were the non-treated group. At 24 h post inoculation, H9N2 viral RNA could be detected at vitelline membrane, embryos, and allantoic fluid of goose embryos from H9N2 inoculated groups. Furthermore, the HA gene of H9N2 virus from vitelline membrane, embryo, allantoic fluid, and gosling shared almost 100% homology with an H9N2 virus isolated from the ovary of breeder goose, which laid these eggs, indicating that H9N2 AIV can be vertically transmitted in goose. The present research study provides evidence that vertical transmission of H9N2 AIV from breeding goose to goslings is possible. Orthomyxoviridae type A that can cause avian infectious diseases  is a member of According to the goose AIV epidemiological investigation in eastern China during 2014–2015, the fertility rate and hatchability of eggs from breeder birds, which were naturally infected with H9N2 AIV, significantly decreased, and H9N2 specific RNA was detected in either breeder birds or in their progenies. Previous research studies have indicated that H5N1 AIV could be vertically transmitted  was originally isolated from dead goose embryos in Shandong Province, China. H9N2-positive dead goose embryo tissue was homogenized in PBS containing antibiotics (20% w/v) and then centrifuged at 6000 g for 30 min at 4°C. The supernatant was then filtered through a 0.22-μm syringe driven filter  and inoculated into 9-day-old SPF chicken eggs through the allantoic cavity route. Inoculated eggs were daily checked, and the allantoic fluid was harvested for virus titration at 72 h post inoculation. The median embryo lethal dose (ELD50) of DP01 strain was tested at 10−7.5/0.2 mL, which was calculated using the Reed and Muench assay  was inoculated through intravenous (group A), intranasal instillation (group B), and throat inoculation (group C) routes. Negative control (group D) was inoculated through intravenous route with PBS. Group E was the non-treated group, which served as the blank control. All groups were separately raised in different animal facilities. The geese were reared on the floor with sterile bedding (wood shavings), where food and water were autoclaved and automatically refilled. All animal experiments were performed according to the guidelines of the Committee on the Ethics of Animals of Shandong and the appropriate biosecurity guidelines, and the protocol was approved by Shandong Agricultural University Animal Care and Use Committee. In addition, all other goose pathogens were tested negative to guarantee our experiment geese had clean background.Because specific pathogen free (SPF) geese were unavailable, 360-day-old healthy Taizhou breeder geese were obtained from the Taizhou breeder goose farm  for bird trail purpose. Firstly, the geese were raised for 20 days to guarantee normal egg production before inoculation. Serum samples and cloacal/tracheal swabs were then collected from breeder geese and tested by hemagglutination inhibition and RT-PCR  samples from dead embryos were collected and stored at −80°C for virus detection and isolation.TransScript® All-in-One First-Strand cDNA Synthesis SuperMix . The primers of H9N2 AIV detection were originally designed according to conserved sequences of H9N2 strains in GenBank  using Trizol reagent  according to the manufacturer's instructions. The extracted RNA was then used for cDNA synthesizing using nk Table . DetectiTransScript® DNA Polymerase High Fidelity  with amplification primers  were normalized per 1 μg of total RNA. Furthermore, qRT-PCR was performed using the SYBR Green PCR kit  in a total volume of 25 μl following the product instructions. The amplification program was set as follows: 95°C for 5 min; 40 cycles of denaturation at 94°C for 15 s and 60°C for 34 s; and a final melting curve analysis step. Each sample was analyzed in triplicates.H9N2 viral RNA concentration was measured from fallopian tube and ovarian tissues in breeder goose infected with H9N2 AIV at 1, 3, 5, and 7 dpi using quantitative SYBR Green I real-time PCR (qRT-PCR) with the 7500 Fast Real-Time PCR System . The primers were designed following conserved sequences of H9N2 strains in GenBank  were compared to the original DP01 isolate. The alignment was constructed using the ClustalW method of MegAlign program of DNA Star software  was used for statistical analysis, and data are expressed as mean ± In 2014–2015, we conducted an AIV epidemiological investigation on goose in eastern China. Egg production drop, sand shell egg, failed capability of embryos to break the shells out Figure , and decApproximately half of the geese in group A showed diarrhea and reduced appetite at 1 dpi, while three dead geese were found at 3 dpi first. In group B, four dead geese were found at 5 dpi, and the most remarkable characteristic was watery eyes and edema that appeared in groups A and B at 9 dpi. In group C, two dead geese were found at 5 dpi, and some geese exhibited slight respiratory distress at 7 dpi; however, approximately half of them showed reduced respiratory distress signs at 9 dpi. No dead geese were found in groups D and E. Importantly, newly hatched goslings exhibited swollen eyelids and watery eyes Figure ; the yolAs shown in Figure RT-PCR amplified products were detected by electrophoresis on a 0.8% agarose gel, and electrophoresis results corresponded with the amplified length of target gene. As shown in Table P < 0.01). Viral RNA was not detected in control groups (data not shown).Quantitative measurements of viral RNA concentration revealed highest levels at 5 dpi, with a slight decrease at 7 dpi  between the DP01 isolate and the SD06 strain , intranasal instillation (group B), and throat inoculation (group C). As shown in Table In the present study, viral RNAs were detected from fallopian tube and ovarian tissues in breeder goose infected with H9N2 AIV at 1, 3, 5, and 7 dpi. Notably, the concentration of viral RNA reached a peak at 5 dpi Figure , 4a,b anH9N2 AIV can be divided into Northern America and Eurasian strains. The Eurasian strains were also divided into three groups as follows: the I lineage represented by A/Duck/Hong Kong/Y280/97 (Y280-like), the II lineage represented by A/Duck/Hong Kong/Y439 (Y439-like), and the III lineage represented by A/Quail/Hong Kong/G1/97 (G1-like; Lin et al., To summarize, our findings provided adequate evidence that H9N2 AIV is vertically transmitted from breeding goose to goslings.GY and AW completed most of the experiments. YT and YD designed experiments and reviewed the manuscript.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.


C. elegans and human mesenchymal stem cells (hMSCs) by disrupting the SKN-1/Nrf2-mediated oxidative stress response. Specifically, TCS exposure affected C. elegans survival and hMSC proliferation in a dose-dependent manner. Cellular analysis showed that TCS inhibited the nuclear localization of SKN-1/Nrf2 and the expression of its target genes, which were associated with oxidative stress response. Notably, TCS-induced toxicity was significantly reduced by either antioxidant treatment or constitutive SKN-1/Nrf2 activation. As Nrf2 is strongly associated with aging and chemoresistance, these findings will provide a novel approach to the identification of therapeutic targets and disease treatment.Triclosan (TCS), an antimicrobial chemical with potential endocrine-disrupting properties, may pose a risk to early embryonic development and cellular homeostasis during adulthood. Here, we show that TCS induces toxicity in both the nematode  Evidence from in vitro and in vivo animal studies has linked TCS to numerous human health problems, including allergies, asthma, and eczema in children, as well as breast cancer and neurodegenerative diseases in adults, through various modes of action4.Triclosan  is a broad-spectrum antimicrobial agent that is commonly used in personal care products  and medical devices + leakage5. Importantly, TCS bears a structural similarity to the thyroid hormone thyroxine (T4) and to other known endocrine disruptors, including polychlorinated biphenyls, diethylstilbestrol, and bisphenol A6. The aromatic nature and high chlorine content of TCS confer resistance to degradation and persistence in the environment4. Similarly, a pharmacokinetic study determined that significant levels of TCS were widely detected in human body fluids and in the surrounding environment4. Recent studies have also shown that TCS may interfere with thyroid hormone metabolism in humans by acting as a potential endocrine-disrupting chemical (EDC)7. TCS was shown to induce thyroid hormone clearance, possibly through the activation of nuclear receptors such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Activated receptors eventually lead to hypothyroxinaemia (significant reduction in serum thyroxine levels) – a hallmark of overt hypothyroidism9. Furthermore, TCS was reported to inhibit the detoxification of foreign chemicals in the body through the interaction with the same nuclear receptors, inducing the expression of hepatic cytochrome P450 2B10 (CYP2b10)10. In vivo studies in mouse models have shown that TCS disrupted liver integrity and compromised liver function10. Moreover, mice subjected to TCS were more susceptible to chemically induced liver tumors10. Based on these investigations and the potential risk associated with TCS exposure, it is currently classified as a category III compound by the FDA1, which has recently banned its use in antibacterial soaps. However, the underlying mechanisms by which TCS causes these numerous health problems at the molecular and cellular levels remain poorly understood.TCS exerts its antimicrobial effect by interfering with enoyl-acyl carrier protein reductase (FabI) activity, which is required for fatty acid and biotin biosynthesis. This ultimately leads to the suppression of bacterial growth. Also, TCS destabilizes bacterial membrane by inducing K11. The antioxidant response elements (AREs) are regulatory DNA sequences common to Nrf2 downstream target genes. Upon induction, Nrf2 dissociates from its cytoplasmic scaffold protein, Keap1, and translocates to the nucleus where it activates a plethora of genes governing energy metabolism, reactive oxygen species (ROS) scavenging and elimination, anti-inflammatory signalling, and xenobiotic detoxification14. SKN-1 (SKiNhead-1) is the C. elegans homolog of Nrf2, and both proteins share the conserved function of cellular defense in response to oxidative stress and xenobiotics16. We here show that TCS induces toxicity, at least in part, by disrupting the SKN-1/Nrf2-mediated oxidative stress response. In this study, we used two powerful model systems, the nematode C. elegans and human mesenchymal stem cells (hMSCs). C. elegans has emerged as an attractive model system for the functional analysis of various bioactive and natural compounds. hMSCs are currently used in many clinical trials and regenerative medicine studies for their multipotency and self-renewal capabilities. Since the mechanism of action of TCS in SKN-1/Nrf2-mediated oxidative stress response may be conserved in diverse cellular processes during animal development, tissue homeostasis, and disease modelling, our findings represent seminal insights on the interaction of TCS with host cells and the role of SKN-1/Nrf2 in xenobiotic stress response.Nuclear factor erythroid-2-related factor 2 (Nrf2) is a mammalian, basic leucine zipper transcription factor involved in the activation of more than 100 genes responsible for cellular stress response and cytoprotectionN,N-diethyl-meta-toluamide (DEET), and methylparaben (MP) , isolated embryos were placed on NGM plates containing 0.2 mM TCS, 0.2 mM DEET, 0.2 mM MP, or 0.2% ethanol (EtOH), which was used as a control, and incubated for 36 hours at 20 °C , MP) Fig. . TCS is  °C Fig. . The sur °C Fig. . Resista °C Fig. . We also °C Fig. . We next °C Fig. . This re °C Fig. . However °C Fig. . Taken t2. Also, levels as high as 2,100 ug/kg lipid weight and 3790 ug/L were detected in human breast milk20, and urine21, respectively. Although the effective concentration of TCS was higher than environmentally relevant levels, in toxicological paradigm, it often requires higher concentrations of drugs to achieve bioavailability. Also, C. elegans has an outer cuticle that protects against the influx of chemicals into the organism23. This renders the nematode more resistant to chemical exposure, necessitating high testing concentrations. Consistent with this notion, mutations in cuticle proteins  cause hypersensitivity to EDCs in C. elegans22. Exposure levels in the study were chosen to facilitate the identification of molecular mechanisms associated with TCS treatment in a relatively short period of time, as opposed to the low concentrations found in the environment which would typically require long, repeated exposure incidents for a biological response to ensue.We also tested the short-term effect of TCS exposure on the survival rate of wild-type worms. L1 synchronized wild-type worms were subjected to TCS and EtOH control in M9 liquid buffer for 1 hour at 20 °C and the survival rates were scored Fig. . NotablyNext, to assess the inter-stage variation in response to TCS, the survival rate of adult worms was also measured. The results showed that TCS decreased the survival of adult worms by a similar magnitude  mutant, which is sensitive to oxidative stress27. L1 stage mev-1(kn1) mutants were exposed to 0–0.4 mM TCS for 1 hour at 20 °C and their survival rates were determined as described above. Compared to wild-type worms, the survival rate of mev-1(kn1) mutants was significantly decreased even at the TCS concentration of 0.1 mM  mutants by H2DCF-DA staining in the absence or presence of TCS. In congruence with the survival rate findings, intracellular ROS levels were significantly increased in the mev-1(kn1) mutants even at the TCS concentration of 0.025 mM  and recorded their survival rates daily under a dissecting microscope. TCS decreased the lifespan of wild-type worms in a dose-dependent manner  mutants to test whether the observed lifespan shortening was related to oxidative stress. Mutations in the mev-1 gene rendered worms hypersensitive to oxidative stress, and led to a slightly decreased lifespan29  mutants was divided by that of wild-type worms at each concentration of TCS. Even at the low concentration of 0.01 mM, the normalized average lifespan of mev-1(kn1) mutants was significantly decreased , while six genes were significantly downregulated ; of these, gcs-1 and pmp-3 were profoundly downregulated following an increase in TCS concentration  and pmp-3(ok1087) single mutant worms. The gene gcs-1, which encodes for γ-glutamyl-cysteine synthetase (γGCS), synthesizes glutathione (GSH) and plays a crucial role in the detoxification of oxidants and toxins in cells33. The gene pmp-3 (encoding an ABC transporter) modulates the absorption, metabolism, and cytotoxicity of pharmacological agents34. L1 stage wild-type, gcs-1 (ok436), and pmp-3 (ok1087) worms were treated with TCS at concentrations of 0, 0.1, 0.2, and 0.4 mM for 1 hour at 20 °C and the survival rates were measured as described above. The survival rate of wild-type worms was approximately 50% and 15% at 0.2 mM and 0.4 mM TCS, respectively  and pmp-3(ok1087) mutants, even at 0.1 mM TCS , and pmp-3(ok1087) worms using the molecular probe H2DCF-DA in the absence or presence of TCS. Notably, ROS levels in gcs-1(RNAi) and pmp-3(ok1087) mutant worms were significantly higher than those in wild-type worms even at the lower concentration (0.025 mM) of TCS  of SKN-1 proteins increases the expression of detoxification and antioxidant-related genes, which reduce cellular oxidative stress38. qRT-PCR analysis revealed that the expression of the skn-1 gene is slightly subverted in response to TCS   gain-of-function (gf) mutant ), which has been shown to activate the expression of genes associated with metabolism, adaptation to starvation, aging, and survival39. The skn-1(gf) mutant worms were exposed to lethal concentrations (≥0.4 mM) of TCS for 1 hour and the survival rates were compared with those of wild-type control worms. The skn-1(gf) mutants, in comparison to wild-type worms, were more resistant to the lethal concentration of TCS  Fig. . We nextTCS Fig.  and exhiTCS Fig. . These fC. elegans, oxidative stress induces the expression of gcs-1, a direct downstream target of SKN-1, in the intestines15. To determine whether TCS inhibits the expression of gcs-1 through SKN-1, we used a transgenic worm that expresses a gcs-1 promoter::gfp transgene15. L1-staged gcs-1::gfp transgenic worms were exposed to SA  and TCS  for 6 hours at 20 °C. Under normal conditions, GCS-1::GFP was found to be detectable in the pharynx, but its expression was very weak in the intestine15. As previously reported15, SA exposure dramatically increased GCS-1::GFP expression in the anterior and posterior regions of C. elegans intestine  that show increased resistance to chemotherapeutic agents41. To test whether GSH supplementation could protect worms against TCS-induced toxicity, wild-type worms were pre-incubated with different concentrations of GSH (0–0.6 mM) for 1 hour at 20 °C, and then exposed to a lethal concentration of TCS (0.4 mM) for an additional 1 hour. Notably, the survival rate was increased by GSH supplementation in a dose-dependent manner -hMSCs were exposed to four different concentrations  of TCS for 7 days and the proliferation rates were measured using an EZ-Cytox kit. No difference in the proliferation rate between control EP-hMSCs and 0.01 and 0.02 mM TCS-treated EP-hMSCs was observed  to explore whether the findings observed in worms were conserved in vertebrates. hMSCs have demonstrated great potential in the development of ved Fig. . Howeverved Fig. . These rNrf2 mRNAs and proteins in hMSCs, EP-hMSCs were treated with 0, 0.01, 0.02, and 0.04 mM TCS for 12 hours. The expression levels of Nrf2 mRNAs were normalized to that of 18 s rRNA. The qRT-PCR results showed that TCS slightly reduced the expression of Nrf2 mRNA at all doses tested  and NQO-1 (encoding NAD(P)H quinone dehydrogenase-1) mRNAs, which are known as downstream target genes of Nrf2, by qRT-PCR. Notably, the expression levels of HO-1 and NQO-1 were significantly decreased by TCS treatment  is an activator of Nrf2. t-BHQ promotes Nrf2 stabilization by preventing binding with Keap147, resulting in a higher hMSC proliferation rate. To test whether TCS inhibited t-BHQ-induced hMSC proliferation rate, cells were treated with 0.05% DMSO control, t-BHQ (0.01 mM), and a t-BHQ (0.01 mM)/TCS (0.02 mM) mixture for 7 days, and the proliferation rates were determined. As we previously reported48, t-BHQ (0.01 mM) treatment enhanced the proliferation rate compared with the DMSO control rol Fig. . Howeverrol Fig. . Next, trol Fig. . Howeverrol Fig. . Next, wase Fig. . These rC. elegans and hMSCs. Based on our findings, we have suggested a possible action mechanism of TCS in SKN-1/Nrf2-mediated oxidative stress response and detoxification , which are associated with oxidative stress response and detoxification mechanisms. GCS-1/γGCS plays an important role in the detoxification of oxidants and toxins from cells33. The PMP-3/ABC transporter mediates the efflux of molecules, including metabolites and toxic agents . However, TCS inhibited the translocation of pSKN-1/pNrf2 proteins into the nucleus and thereby suppressed the expression of the gcs-1/γGCS1 and pmp-3/ABC transporter genes. Therefore, we suggest that TCS induces oxidative stress and intracellular accumulation of toxins, at least in part, through disrupting SKN-1/Nrf2-mediated oxidative stress response and detoxification mechanism.This work demonstrated that TCS induced oxidative stress by the disruption of SKN-1/Nrf2-mediated oxidative stress response, in two powerful model systems, ion Fig. . Under o52. Moreover, ROS plays a major role in tumor initiation and survival, as induced by a variety of agents in several model systems, including humans. ROS-induced oxidative stress can be reduced by the main cellular defense mechanisms53. One of the major regulators is Nrf2, which activates the cellular antioxidant response by inducing the transcription of its target genes. Nrf2 target genes protect cells from the effects of xenobiotics and oxidative stress54 and inhibit carcinogenesis55. Therefore, although Nrf2 has been generally considered as a tumor suppressor, it can also protect cancer cells from oxidative stress and chemotherapeutic agents  and their elimination by protective mechanisms. This imbalance leads to the oxidative damage of biomolecules and cells within the body. Oxidative stress intertwine with the multiple stages of the cancer process, including initiation, promotion, and progression57. The results in C. elegans and hMSCs indicated that TCS may induce cellular toxicity by the disruption of SKN-1/Nrf2-mediated oxidative stress response. Specifically, TCS inhibited the nuclear localization of Nrf2 in EP-hMSCs, and consequently, the expression of its downstream target genes . These results suggest that TCS may promote the senescence of EP-hMSCs. It was recently reported that Nrf2 activation by t-BHQ promotes stem cell capacity in late passage (LP)-hMSCs through Sirt1 expression48. After stimulation by t-BHQ, Nrf2 proteins are released from Keap1 (Kelch-like ECH-Associated Protein 1) and translocate into the nucleus58. t-BHQ treatment in LP-MSCs promotes Nrf2 phosphorylation, which activates the expression of target genes (HO-1 and NQO1) and Sirt1 by similar levels to those observed in EP-hMSCs. Intriguingly, t-BHQ-induced activation of Nrf2 in LP-hMSCs promoted stem cell capacity of hMSCs with increased proliferation and differentiation abilities into osteogenic and adipogenic lineages. These results suggest that active Nrf2 may mediate Sirt1 to regain stem cell capacity in LP-hMSCs. As TCS affected Nrf2 nuclear localization, we proposed that TCS may also affect stem cell capacity during aging. Altogether, since Nrf2 pathways are involved in multiple physiological processes during development and in cancer cell chemoresistance, our findings provide a paradigm that may facilitate pharmacological approaches to therapeutic targeting and disease modelling.Various environmental stresses can induce the excessive production of ROS, which triggers cellular senescence and abnormal differentiation in hMSCsN,N-diethyl-meta-toluamide), and methylparaben  as shown in Fig. Triclosan  phenol, TCS), DEET , 1 ml Cholesterol (5 mg/1 ml), H2O to 1 litre) media before plating   and mixtures of TCS, DEET, and MP (0.2 mM) of each were prepared by adding appropriate quantities of solutes to a volumetric flask and filling to the mark with ethanol.TCS, DEET, and MP powders were dissolved in ethanol as a solvent. To investigate the toxicity of selected chemicals, all stock solutions (100 mM) were diluted to final concentrations of 0.1, 0.2, and 0.4 mM with the sterilized NGM . Then, the obtained embryos or L1 stage larvae were exposed to the solution consisting of either only TCS or a combination of TCS with DEET and MP for 1 hour at 20 °C, and the effects of the chemicals on survival rate, lifespan, and gene expression were measured.Wild-type N2, other mutants and transgenic worms were obtained from the Caenorhabditis Genetics Center (CGC) (see Supplementary Table mev-1(kn1), gcs-1(ok436), and pmp-3(ok1087) mutants  and three mev-1(kn1) mutants. Synchronized L1 stage larvae were transferred to the NGM plate in the absence or presence of TCS (0.02 mM and 0.04 mM). The worms were transferred to fresh NGM plate every 2 days and dead worms were counted daily. Standard deviation bars were calculated from three independent experiments.The lifespan assays were performed using wild-type(N2) and 2DCF-DA). Wild-type(N2) or mutant worms were exposed to either EtOH or TCS for the measurement of intracellular ROS. Chemical-treated L1-arrested larvae were then transferred into the 96-well plate containing 50 μL of M9 buffer. Immediately 50 μL of 50 μM H2DCF-DA was added to each well. The basal fluorescence was quantified using a microplate fluorescence reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Plates were read every 30 minutes for 5 hours. Three replicate experiments were conducted.Intracellular ROS in the worms was measured using molecular probe 2′,7′-dichlorodihydrofluoroscein diacetate . Quality and purity of total RNA was quantified by a Nanodrop ND-1000 . For reverse transcription (RT), 650 ng of total RNA from control and each treatment sample were used. RNA was transcribed to single-stranded cDNA using the reverse primer Poly(dT). RT-PCR was conducted in 250 μL microcentrifuge tubes and reacted in a 15 μL solution. This solution consists of precalculated amounts of DNase/RNase-free water, 1,000 ng of total RNA, 0.19 μL RNase inhibitor (20 U/μL), 0.15 μL 100 mM dNTPs, 10× RT buffer, 1 μL MultiScribeTM reverse transcriptase (50 U/μL), and 2 μL of Poly(dT) primer mix. The reactions were performed using an Eppendorf Mastercycler® Pro PCR machine. Quantitative real-time PCR (qRT-PCR) reactions were performed in 10 μL solutions, consisting of 2 μL DNase/RNase free water, 5 μL SYBR green PCR master mix, 1 μL RT PCR product (diluted with 85 μL of DNase/RNase free water), and 2 μL of primer mix. The reactions were performed with a VIIATM 7 Real-Time PCR system using programmed temperature. The programmed temperature evaluation starts with an initial polymerase activation stage for 10 minutes at 95 °C, followed by denaturation for 15 seconds at 95 °C, and an annealing/extension step for 60 seconds at 60 °C. The latter two steps were repeated for 40 cycles. Four biological replicates were performed per treatment group with each having three technical replicates. In qRT-PCR, the expression was normalized to tba-1, which encodes α-tubulin, and 18S rRNA61.After the exposure of L1 stage larva to 0.02 mM and 0.04 mM of TCS, about 2,000 L1 stage larvae were transferred to fresh NGM plates seeded with Wild-type L1 worms were pretreated with GSH (0–0.6 mM) in M9 buffer for 1 hour and then were treated with 0.4 mM TCS for additional 1 hour. Worms were washed three times with M9 buffer and then transferred to NGM agar plates. Their viability was measured under a dissecting microscope. Standard deviation bars were calculated from at least three independent experiments.skn-1::gfp transgenic worms were exposed to 5 mM sodium arsenite for 30 minutes at 20 °C in the absence or the presence of TCS (0.1–1 mM), then covered with a glass slip and examined by fluorescence microscopy. These worms were scored for the presence of SKN-1::GFP in intestinal nuclei 5 minutes later under a fluorescence microscope.To analyze the effect of TCS on the localization of SKN-1::GFP in response to oxidative stress, ldIs3 [gcs-1p::GFP + rol-6(su1006)]) worms were exposed to 0.2 mM sodium arsenite for 6 hours at 20 °C in the absence or presence of TCS (0–0.2 mM). The GCS-1::GFP expression levels were examined under a fluorescence microscope.To analyze the induction of GCS-1::GFP expression in the absence or presence of TCS under oxidative stress condition, LD1171 , and then 1 mL growth media containing 20 μl of EZ-Cytox  solution was added to each well and incubated at 37 °C for 3 hours. Then, the conditioned media were transferred into 96-well plates, and the absorbance was measured at 450 nm using spectrophotometry. All sample were tested in triplicate (n = 3).Cell cytotoxicity for TCS was examined using an EZ-Cytox Kit . TCS-treated MSCs were seeded in 12-well cell culture plates 1 × 10http://www.bioneer.co.kr/). The primers used were as follows: Nrf2 [P164742], HO-1 [P133045], and NQO-1 [P113225]. 18S rRNA was used as an internal control and the sequence was as follows: 5′-acacggacaggattgacagattg-3′  and 5′-gccagagtctcgttcgttatcg-3′ (antisense). Mean cycle threshold values from triplicate (n = 3) measurements were used to calculate gene expression, with normalization to 18S rRNA levels.RNA was isolated using an RNeasy kit . The isolated RNAs were then reverse-transcribed using an Omniscript kit (Qiagen). Primer sets were validated and purchased from Bioneer  according to the manufacturer’s instructions. The nuclear and cytosolic proteins were analyzed by western blot analysis.Ethanol- or TCS-treated hMSCs were lysed in passive lysis buffer  after 12 hours of TCS treatment. The Bio-Rad protein assay  were used to determine protein concentrations, and 10 μg of proteins were analyzed by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel (Sigma). 5% skim milk  was used to block the transferred membranes, and the membranes were incubated for overnight with antibodies against Nrf2 , phosphorylated-Nrf2 , LAMIN-B (Santa Cruz Biotechnology), and LDH (Santa Cruz Biotechnology). The membranes were further probed with an antibody against β-ACTIN (Santa Cruz Biotechnology), which served as a loading control. See Supplementary Table 2 incubator at 37 °C. The cells were fixed with 4% paraformaldehyde (Sigma) for 30 minutes, and then permeabilized with 1% Triton X-100 for 10 minutes followed by blocking for 1 hour with 5% bovine serum albumin (BSA) in PBS. The cells were incubated with 1:100 dilution of antibodies against Nrf2 (Santa Cruz Biotechnology) for 4 hours at room temperature, and then incubated fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Santa Cruz Biotechnology) at a 1:5,000 dilution in 1% BSA-containing PBS for 1 hour at room temperature in the dark. The nuclei were stained with 4,6-diamidino-2-phenyindole (DAPI) (Sigma) and then examined using a Zeiss LSM700 scanning laser confocal microscope .Ethanol- or TCS-treated hMSCs were seeded at 2,000 cells per square centimeter on four-well glass chamber slides , and the cells were incubated in a 5% COThe data from the survival rate analysis and lifespan assay were plotted using Microsoft Excel software and statistical significance was analyzed by log-rank test. Other data were presented as mean ± standard deviation or standard error of the mean depends on the experiments as indicated. Statistical significance of differences between the control and treated groups were analyzed by one-way analysis of variance (ANOVA). The error bars reflect respective standard deviation values.Supplementary Info