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OBJECTIVES Aortic stenosis or regurgitation that requires operations in children often results from unicuspid valve morphology. In all paediatric patients with this anomaly, we have performed unicuspid valve repair by bicuspidization, creating a new commissure via adding patch material. This study reviewed our experience with this procedure. METHODS All patients with a unicuspid aortic valve who underwent bicuspidization at ≤18 years of age between 2003 and 2018 were evaluated. Autologous pericardium had initially been used for cusp augmentation. Since 2014, decellularized xenogeneic tissue or expanded polytetrafluoroethylene membrane has been applied. RESULTS There were 60 consecutive patients. The median operative age was 13 (1–18) years. Thirty patients had prior surgical or catheter valvuloplasties. Aortic regurgitation, aortic stenosis and their combination were present in 22, 11 and 27 patients, respectively. Autologous pericardium decellularized tissue and expanded polytetrafluoroethylene were used in 45, 11 and 4 patients, respectively, without intraoperative conversion to valve replacement. The overall survival was 96% at 10 years with 2 late deaths. Twenty patients underwent aortic valve reoperation due to patch degeneration (n = 11), suture dehiscence of the patch (n = 3), subaortic stenosis (n = 3) or other reasons (n = 3). Freedom from aortic valve reoperation was 73% and 50% at 5 and 10 years, respectively. Fifteen patients (25%) required valve replacement with pulmonary autograft (n = 14) or prosthesis (n = 1) 5.2 (0.2–13) years after bicuspidization at the age of 19 (10–32) years. CONCLUSIONS Bicuspidization is a safe and reproducible alternative to valve replacement with acceptable freedom from reoperation. It can serve as a bridge to valve replacement providing superior outcomes in adults.

Aortic valve-sparing operations are meant to stabilize the root in order to provide stable aortic valve function. The possibility of graft dilatation has been known in other forms of aortic replacement but is largely ignored in valve-preserving surgery. We describe graft dilatation as a cause of failure late after aortic valve reimplantation. It was corrected by applying the concepts of aortic valve repair.

OBJECTIVE: To assess the feasibility and accuracy of seizure detection based on heart rate variability (HRV) using a wearable electrocardiography (ECG) device. Noninvasive devices for detection of convulsive seizures (generalized tonic-clonic and focal to bilateral tonic-clonic seizures) have been validated in phase 2 and 3 studies. However, detection of nonconvulsive seizures still needs further research, since currently available methods have either low sensitivity or an extremely high false alarm rate (FAR). METHODS: In this phase 2 study, we prospectively recruited patients admitted to long-term video-EEG monitoring (LTM). ECG was recorded using a dedicated wearable device. Seizures were automatically detected using HRV parameters computed off-line, blinded to all other data. We compared the performance of 26 automated algorithms with the seizure time-points marked by experts who reviewed the LTM recording. Patients were classified as responders if >66% of their seizures were detected. RESULTS: We recruited 100 consecutive patients and analyzed 126 seizures (108 nonconvulsive and 18 convulsive) from 43 patients who had seizures during monitoring. The best-performing HRV algorithm combined a measure of sympathetic activity with a measure of how quickly HR changes occurred. The algorithm identified 53.5% of the patients with seizures as responders. Among responders, detection sensitivity was 93.1% (95% CI: 86.6%-99.6%) for all seizures and 90.5% (95% CI: 77.4%-97.3%) for nonconvulsive seizures. FAR was 1.0/24 h (0.11/night). Median seizure detection latency was 30 s. Typically, patients with prominent autonomic nervous system changes were responders: An ictal change of >50 heartbeats per minute predicted who would be responder with a positive predictive value of 87% and a negative predictive value of 90%. SIGNIFICANCE: The automated HRV algorithm, using ECG recorded with a wearable device, has high sensitivity for detecting seizures, including the nonconvulsive ones. FAR was low during the night. This approach is feasible in patients with prominent ictal autonomic changes.

Aim: Adipose tissue-derived microvascular fragments (ad-MVF) are vascularization units for regenerative medicine. We investigated whether University of Wisconsin (UW) solution is suitable for their xeno-free storage. Materials & methods: Murine ad-MVF were cultivated for 24 h in 4°C or 20°C UW solution and 20°C endothelial cell growth medium (control). The ad-MVF were seeded onto collagen–glycosaminoglycan scaffolds, which were analyzed in dorsal skinfold chambers by intravital fluorescence microscopy and histology. Results: All implants exhibited microvascular networks on day 14 with the highest functional microvessel density in controls. Ad-MVF cultivation in UW solution at 4°C resulted in an improved scaffold vascularization compared with cultivation at 20°C. Conclusion: UW solution is suitable for the hypothermic storage of ad-MVF.

Adipose tissue-derived microvascular fragments (ad-MVFs) are promising vascularization units for tissue engineering. In this study, we analysed the effects of normothermic (37°C) and subnormothermic (20°C) short-term cultivation on their viability and network forming capacity. Ad-MVFs from green fluorescent protein (GFP)+ and GFP- C57BL/6 mice were cultivated for 24 hr at 37°C or 20°C. Freshly isolated, noncultivated ad-MVFs served as controls. Number, length, viability, proliferation, and angiogenic activity of the ad-MVFs were assessed by microscopic analysis and proteome profiling. GFP+ ad-MVFs were seeded onto collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of GFP- mice to analyse their vascularization by means of intravital fluorescence microscopy, histology, and immunohistochemistry. Depending on the temperature, short-term cultivation of ad-MVFs markedly changed their expression of multiple proangiogenic and antiangiogenic factors. Moreover, cultivation at 37°C significantly increased the number of apoptotic cells within ad-MVFs, whereas 20°C preserved the viability of ad-MVFs and even promoted the proliferation of endothelial and perivascular cells. Accordingly, ad-MVFs cultivated at 20°C also exhibited an enhanced in vivo vascularization capacity when compared with normothermically cultivated ad-MVFs and noncultivated controls. This was indicated by an accelerated network formation, an increased microvascular remodelling, and a higher density of GFP+ microvessels within implanted matrices. Thus, if ad-MVFs require short-term storage before in vivo application, subnormothermic cultivation should be preferred to normothermic cultivation.

The seeding of tissue constructs with adipose tissue-derived microvascular fragments (ad-MVF) is an emerging pre-vascularisation strategy. Ad-MVF rapidly reassemble into new microvascular networks after in vivo implantation. Herein it was analysed whether this process was improved by erythropoietin (EPO). Ad-MVF were isolated from green fluorescent protein (GFP)+ as well as wild-type C57BL/6 mice and cultivated for 24 h in medium supplemented with EPO (20 IU/mL) or vehicle. Freshly isolated, non-cultivated ad-MVF served as controls. Protein expression, cell viability and proliferation of ad-MVF were assessed by proteome profiler array and fluorescence microscopy. GFP+ ad-MVF were seeded on collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of C57BL/6 mice, to analyse their vascularisation over 14 d by intravital fluorescence microscopy, histology and immunohistochemistry. Cultivation up-regulated the expression of pro- and anti-angiogenic factors within both vehicle- and EPO-treated ad-MVF when compared with non-cultivated controls. Moreover, EPO treatment suppressed cultivation-associated apoptosis and significantly increased the number of proliferating endothelial cells in ad-MVF when compared with vehicle-treated and non-cultivated ad-MVF. Accordingly, implanted matrices seeded with EPO-treated ad-MVF exhibited an improved vascularisation, as indicated by a significantly higher functional microvessel density. The matrices of the three groups contained a comparably large fraction of GFP+ microvessels originating from the ad-MVF, whereas the tissue surrounding the matrices seeded with EPO-treated ad-MVF exhibited a significantly increased microvessel density when compared with the other two groups. These findings indicated that EPO represents a promising cytokine to further boost the excellent vascularisation properties of ad-MVF in tissue-engineering applications.

Adipose tissue-derived microvascular fragments (ad-MVF) are effective vascularization units for tissue engineering. They rapidly reassemble into new microvascular networks after seeding on scaffolds and subsequent in vivo implantation. Herein, we analyzed whether the vascularization capacity of ad-MVF is affected by cryopreservation. Ad-MVF were isolated from the epididymal fat pads of C57BL/6 mice and cryopreserved for 7 days to compare their morphology, viability, cellular composition, and protein expression with freshly isolated control ad-MVF. Moreover, cryopreserved and control ad-MVF from green fluorescent protein (GFP)+ donor mice were seeded on collagen-glycosaminoglycan matrices (Integra® ), which were implanted into dorsal skinfold chambers of GFP- recipient animals to study their vascularization and incorporation using intravital fluorescence microscopy, histology, and immunohistochemistry. Cryopreservation of ad-MVF did not affect vessel morphology and cellular composition. However, cryopreservation was associated with an increased rate of necrotic cells and a significantly reduced number of transplantable ad-MVF. This was compensated by a higher angiogenic activity of the remaining ad-MVF, as indicated by significantly elevated expression levels of pro-angiogenic factors when compared to controls. Accordingly, cryopreserved and control ad-MVF induced a comparable vascularization and incorporation of implanted Integra® without differences in microvascular network formation, maturation, and remodeling. Enhanced angiogenic sprouting even resulted in a higher fraction of GFP+ microvessels within the implants of the cryopreservation group. These findings indicate that cryopreservation of ad-MVF is feasible and, thus, offers the exciting opportunity to build up stocks of readily available vascularization units for future tissue engineering applications.

OBJECTIVES Some studies have suggested that root remodelling is unsuitable as valve-sparing aortic root replacement in children because of the increased risk of valve failure. This study reviewed our experience with root remodelling in children. METHODS All patients who underwent root remodelling at the age of ≤18 years between 1999 and 2016 were evaluated. In 2004, cusp effective height was measured intraoperatively and prolapse was corrected with central plication on the cusp. Suture annuloplasty was introduced after 2009 for annular dilatation. RESULTS There were 17 consecutive patients. The median age at operation was 14 (2.8–18) years. Aortic valve morphology was tricuspid in 10 patients, bicuspid in 5 patients, unicuspid in 1 patient and a pulmonary autograft in 1 patient. Marfan syndrome, Loeys-Dietz syndrome and other connective tissue diseases were present in 11, 1 and 2 patients, respectively. Five patients had more than moderate aortic regurgitation. The median graft size used for root remodelling was 24 (18–26) mm. Cusp repair and annuloplasty were performed in 15 and 14 patients, respectively. The mean follow-up time was 6.5 ± 4.3 years. One patient with preoperatively severely depressed ventricular function died in the hospital from persistent heart failure. One patient (operated on before 2004) required aortic valve reoperation due to cusp prolapse. One patient with a unicuspid valve had developed moderate aortic regurgitation, and the other 14 patients had mild or less regurgitation. The median diameter of the sinus of Valsalva at the last follow-up was 36 (30–43) mm, Z-score of 1.5 (−3.5 to 3.9). CONCLUSIONS Root remodelling can be performed in children with favourable results by appropriate cusp repair and annuloplasty.

We describe a root remodeling technique for the unicuspid and unicommissural aortic valve with a moderately hypoplastic anterior commissure. This technique achieves symmetric bicuspidization without patch insertion to cusps. Partial cusp fusion of the anterior commissure is divided, and two symmetric tongues of a Dacron graft are sutured to each center of cusp insertion lines. The cusp nadirs are relocated posteriorly, and the bicuspidized valve has two sufficiently high commissures from a new angulated virtual basal ring. To optimize valve configuration, annuloplasty and prolapse correction according to effective height concept are added. This technique is a promising option in select patients.

Meal delivery platforms like Uber Eats shape the landscape in cities around the world. This paper addresses forecasting demand on a grid into the short-term future, enabling, for example, predictive routing applications. We propose an approach incorporating both classical forecasting and machine learning methods and adapt model evaluation and selection to typical demand: intermittent with a double-seasonal pattern. An empirical study shows that an exponential smoothing based method trained on past demand data alone achieves optimal accuracy, if at least two months are on record. With a more limited demand history, machine learning is shown to yield more accurate prediction results than classical methods.

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder, characterized by cafe-au-lait macules, benign neurofibromas as well as malignant peripheral nerve sheath tumours, freckling in the axillary or inguinal regions, optic glioma and Lisch nodules (iris hamartomas) and further manifestations like bone deformities etc. Additionally, NF1 patients are at increased risk of early-onset cerebrovascular diseases, the pathogenesis of which has not been clarified yet. Here we report the first case of two siblings with NF1 who suffered an acute ischemic stroke. Professionals treating NF1 patients should be aware of the elevated risk of stroke in this population. Large prospective studies are needed to establish optimal guidelines for diagnosis, monitoring and treatment of cerebrovascular disease in patients suffering from NF1, as well as to achieve a consensus on routine vascular screening in NF1.

Introduction We examined the role of ultrasound in diagnosing and localizing ulnar neuropathy (UN) at the elbow. Methods Forty-one patients referred for UN and 43 healthy subjects were included. Cross-sectional area (CSA) of the ulnar nerve was measured by ultrasound at 5 locations across the elbow and at the wrist. Patients underwent near-nerve conduction studies (N-NCS). Results Ultrasound (US) had a sensitivity of 80.5%, N-NCS had a sensitivity of 70.7%, and combined sensitivity was 85.4% in diagnosing UN. Using N-NCS as a reference standard, US reached a sensitivity of 93.1% and a specificity of 50.0% in diagnosing UN, and a sensitivity of 77.3% and a specificity of 42.9% in localizing UN at the elbow. There was a reduction in mean CSA at 3-month follow-up. Conclusions N-NCS and US are complementary methods and have the potential to increase the sensitivity for diagnosing and localizing UN at the elbow. Muscle Nerve 52: 759–766, 2015

Background Routine quantitative assessment of left ventricular (LV) volumes with echocardiography is hindered by time-consuming methods requiring a manual trace of the LV cavity from two apical two-dimensional planes. Thus, the aim of this study was to evaluate faster new semiautomatic echocardiographic methods that could represent a feasible alternative for the assessment of LV volumes and ejection fraction (EF) in clinical practice. Methods Two semiautomatic methods, the automated EF (Auto-EF) for two-dimensional echocardiography and the 4D Auto LVQ tool for three-dimensional echocardiography (3DE), were compared with the biplane modified Simpson's method and cardiac magnetic resonance (CMR) imaging in 47 patients. To evaluate the accuracy of volumetry, additional in vitro measurements using water-filled latex balloons were performed with both modalities. Results Results of balloon volumetry by echocardiography and CMR measurements were in good agreement with real balloon volumes. The mean LV EF was 45 ± 11% by Auto-EF, 45 ± 11% by 3DE, 48 ± 11% by Simpson's method, and 54 ± 12% by CMR. Linear regression and Bland-Altman analyses showed good associations for semiautomatic methods with Simpson's method (Auto-EF, r = 0.85, bias = 3%, limits of agreement [LOA] = 12%; 3DE, r = 0.79, bias = 3%, LOA = 14%), as well as with CMR (Auto-EF, r = 0.74, bias = 9%, LOA = 17%; 3DE, r = 0.73, bias = 9%, LOA = 17%). Intra- and interobserver variability were 6% and 12% with Auto-EF and 8% and 11% with 3DE, respectively. Conclusions Good correlations between semiautomatic echocardiographic parameters for assessment of LV volumes and EF could be observed when compared with Simpson's method or CMR. However, intertechnique agreement analysis of absolute LV volumes revealed considerable differences, with significant underestimation of volumes and EF with respect to CMR.

Purpose To compare the value of Dobutamine stress echocardiography (DSE) with that provided by Dobutamine Cardiac Magnetic Resonance (DCMR) for the non-invasive risk stratification of patients with suspected or known coronary artery disease (CAD). Methods Patients with suspected or known CAD underwent either DSE or DCMR using the same standardized protocol. Patient matching was then performed retrospectively for age, gender and risk factors. Outcome data including cardiac death and non-fatal myocardial infarction (defined as hard cardiac events) and ‘late' revascularization performed >90days after the diagnostic procedures were collected during at least 6months. Results Follow-up data were available in 1852 patients who completed either DSE (n=884) or DCMR (n=884) during a mean follow-up duration of 4.1±2.4 and 3.9±1.9years, respectively (p=NS). Matched patients exhibited an overall high risk profile (69±9years; 69% male, 70% history of CAD and 26% diabetes mellitus in both groups). Using multivariable analysis, both modalities successfully identified patients with inducible ischemia at higher risk for subsequent hard cardiac events, surpassing the value of conventional risk factors like age, male gender and diabetes (HR=9.2; 95%CI=5.6–14.9 for DCMR versus 2.5; 95%CI=1.7–3.7 for DSE). By testing for interaction the predictive capacity of DCMR was higher than that provided by DSE (p=0.02). Patients with negative DCMR exhibited lower event rates compared to those with negative DSE (annual hard cardiac event rate of 0.8% versus 3.2%, p=0.002). Conclusions DSE & DCMR aid the risk stratification of CAD patients. However, inducible WMA during DCMR are associated with a higher risk for subsequent cardiac events. Patients with negative DCMR on the other hand, exhibited a lower event rate compared to those with negative DSE.

Background So far, only generalized tonic-clonic seizures can be reliably detected with non-invasive wearable devices. We aimed to develop an automated seizure detection algorithm using a wearable ECG-device for detecting both GTC and focal seizures. Material and methods We recorded ECG using a dedicated wearable device (ePatch®) during long-term video-EEG monitoring. In this phase-2 clinical study, 100 patients were prospectively recruited; 43 of the patients had 126 seizures (108 focal, 18 GTC) of >20 s duration during recording (941 h training data, 2238 h test data). We analyzed 20 heart rate variability (HRV)-parameters and 6 combinations of these using either 50 or 100 R-R intervals sliding window with maximum overlapping. Each HRV-parameters cut-off value for seizure-alarm was set to 105% of the highest non-seizure period during training data of the same patient. Positive responders of seizure detection were defined, for each HRV-parameter, as patients with >66% of seizures detected. Results In total, 53.5% of the patients were responders for the best performing algorithm. In these patients, the method achieved a sensitivity of 93.1% and false detection rate of 1.1/day. An average of >50 beats/minute HR increase or decrease during seizure(s) is a positive predictor of being a responder of seizure detection (PPV: 87.0%, NPV: 90.0%), making it easy to define for which patients a reliable seizure alarm is feasible. Conclusions High sensitivity and low false positive alarm rates can be achieved with our algorithm analyzing ECG-signals using the wearable device in persons with average HR changes >50 beats/min during seizures.

BACKGROUND: Several EEG criteria have been proposed for diagnosis of non-convulsive status epilepticus (NCSE), but none have been clinically validated. We aimed to assess the diagnostic accuracy of the EEG criteria proposed by a panel of experts at the fourth London-Innsbruck Colloquium on Status Epilepticus in Salzburg, 2013 (henceforth called the Salzburg criteria).METHODS: We did a retrospective, diagnostic accuracy study using EEG recordings from patients admitted for neurological symptoms or signs to three centres in two countries (Danish Epilepsy Centre, Dianalund, Denmark; Aarhus University Hospital, Aarhus, Denmark; and Paracelsus Medical University, Salzburg, Austria). Participants were included from the Danish centres if they were aged 4 months or older, and from the Austrian centre if aged 18 years or older. Participants were sorted into two groups: consecutive patients under clinical suspicion of having NCSE (the clinical validation group) or consecutive patients with abnormal EEG findings but no clinical suspicion of NCSE (the control group). Two raters blinded to all other patient data retrospectively analysed the EEG recordings and, using the Salzburg criteria, categorised patients as in NCSE or not in NCSE. By comparing with a reference standard inferred from all clinical and para-clinical data, therapeutic response, and the final outcome, we calculated sensitivity, specificity, overall diagnostic accuracy, positive and negative predictive values, and inter-rater agreement for the Salzburg criteria. The reference standard was inferred by two raters who were blinded to the scorings of the Salzburg criteria.FINDINGS: We retrospectively reviewed EEG data from 220 patients. EEGs in the clinical validation group were recorded in 120 patients between Jan 1, and Feb 28, 2014 (Austria), and Aug 1, 2014, and Jan 31, 2015 (Denmark). EEGs in the control group were recorded in 100 patients between Jan 13 and Jan 22, 2014 (Austria) and Jan 12 and Jan 26, 2015 (Denmark). According to the reference standard, 43 (36%) of the 120 patients in the validation group had NCSE. In the validation cohort sensitivity was 97·7% (95% CI 87·9-99·6) and specificity was 89·6% (80·8-94·6); overall accuracy was 92·5% (88·3-97·5). Positive predictive value was 84·0% (95% CI 74·1-91·5) and negative predictive value was 98·6% (94·4-100). Three people in the control group (n=100) fulfilled the Salzburg criteria and were therefore false positives (specificity 97·0%, 95% CI 91·5-99·0; sensitivity not calculable). Inter-rater agreement was high for both the Salzburg criteria (k=0·87) and for the reference standard (k=0·95). Therapeutic changes occurred significantly more often in the group of patients fulfilling Salzburg criteria (42 [84%] of 50 patients) than in those who did not (11 [16%] of 70; pINTERPRETATION: The Salzburg criteria for diagnosis of NCSE have high diagnostic accuracy and excellent inter-rater agreement, making them suitable for implementation in clinical practice.FUNDING: None.

Z-disks, the mechanical integration sites of heart and skeletal muscle cells, link anchorage of myofilaments to force reception and processing. The key molecules that enable the Z-disk to persistently withstand the extreme mechanical forces during muscle contraction have not yet been identified. Here we isolated nexilin (encoded by NEXN) as a novel Z-disk protein. Loss of nexilin in zebrafish led to perturbed Z-disk stability and heart failure. To evaluate the role of nexilin in human heart failure, we performed a genetic association study on individuals with dilated cardiomyopathy and found several mutations in NEXN associated with the disease. Nexilin mutation carriers showed the same cardiac Z-disk pathology as observed in nexilin-deficient zebrafish. Expression in zebrafish of nexilin proteins encoded by NEXN mutant alleles induced Z-disk damage and heart failure, demonstrating a dominant-negative effect and confirming the disease-causing nature of these mutations. Increasing mechanical strain aggravated Z-disk damage in nexilin-deficient skeletal muscle, implying a unique role of nexilin in protecting Z-disks from mechanical trauma.

Although it is well known that mutations in the cardiac essential myosin light chain-1 ( cmlc-1 ) gene can cause hypertrophic cardiomyopathy, the precise in vivo structural and functional roles of cMLC-1 in the heart are only poorly understood. We have isolated the zebrafish mutant lazy susan ( laz ), which displays severely reduced contractility of both heart chambers. By positional cloning, we identified a nonsense mutation within the zebrafish cmlc-1 gene to be responsible for the laz phenotype, leading to expression of a carboxyl-terminally truncated cMLC-1. Whereas complete loss of cMLC-1 leads to cardiac acontractility attributable to impaired cardiac sarcomerogenesis, expression of a carboxyl-terminally truncated cMLC-1 in laz mutant hearts is sufficient for normal cardiac sarcomerogenesis but severely impairs cardiac contractility in a cell-autonomous fashion. Whereas overexpression of wild-type cMLC-1 restores contractility of laz mutant cardiomyocytes, overexpression of phosphorylation site serine 195–deficient cMLC-1 (cMLC-1 S195A ) does not reconstitute cardiac contractility in laz mutant cardiomyocytes. By contrast, introduction of a phosphomimetic amino acid on position 195 (cMLC-1 S195D ) rescues cardiomyocyte contractility, demonstrating for the first time an essential role of the carboxyl terminus and especially of serine 195 of cMLC-1 in the regulation of cardiac contractility.

In this study, the temporal shape of voice-induced nitric oxide (NO) signals in exhaled air has been investigated in eight healthy individuals by means of laser magnetic resonance spectroscopy. The results of the experimental part have been compared with calculated signals obtained by using a simple one-compartment model of the paranasal sinuses. In the experimental part, a rapidly increasing NO concentration has been found when the subjects started humming. After reaching a maximum, the emission starts to decrease with the shape of an exponential decay and finally reaches a constant level. The time constant of this decay (NO washout) is 3.0 ± 1.2 s. The peak height of the NO emission during humming increases when the time between two humming processes increases. When no voice-induced NO emission takes place, the NO concentration in the paranasal sinuses rebuilds again to a maximum concentration. The typical time constant for the NO recovery is 4.5 ± 3.2 min. A three-compartment model defining exactly the geometry and anatomy of the paranasal sinuses has been developed that is based on three main assumptions of the NO dynamics: 1) constant NO production of the epithelium in the sinuses; 2) the rate of the chemical reaction of NO with the epithelium of the paranasal sinuses is proportional to the NO concentration; and 3) the emission of NO from the sinuses (volume/s) is proportional to the NO concentration. It is shown that the three-compartment model under the experimental conditions can be reduced to a one-compartment model, which describes the complete temporal behavior of the NO exchange.

Recent ethical and moral lapses, defined as decisions or actions not in line with the Air Force Core Values, are not isolated occurrences. Rather, they are punctuated events on a spectrum of behavior. This spectrum exists in a complex culture with competing and conflicting values and interests that can create a moral dilemma for Airmen. Positive and negative behavioral drivers determine the choices of an individual who is faced with such a dilemma. An organization can also influence the individual's decision-making through separate negative drivers collectively referred to as moral gravity. Lapses in an individual's commitment to the Air Force Core Values occur when positive individual drivers are not emphasized in the presence of moral gravity. The purpose of this report is to identify specific actions that will eliminate negative drivers and replicate positive drivers, and thus promote behavior and decision-making more in line with the Core Values.

Immunohistochemical investigations of the guinea pig cochlea, using a specific antibody to the inducible isoform of NO synthase (iNOS/NOS II), have been performed 3 weeks after closure of the right endolymphatic duct (n=7). Endolymphatic hydrops, the morphological substrate of Meniere's disease, became evident by distension of the Reissner's membrane. iNOS expression could be noted in endothelium, spiral ganglion cells, in nerve fibers, in supporting cells of the organ of Corti and cells of the spiral ligament. Temporal bones of non-operated controls (n=6) as well as of sham-operated animals (n=3) did not show structures positive to iNOS. These findings imply that iNOS-generated NO could be involved in the pathophysiology of cochlear dysfunction in Meniere's disease.

Integration of tactile afferent signals with motor commands is crucial for the performance of purposeful movements such as during manipulation of an object in the hand. To study the somatotopic organization of sensorimotor integration we applied electrical peripheral conditioning stimuli to a digit located near (homotopic stimulation) or distant from (heterotopic stimulation) relaxed or isometrically contracted intrinsic hand muscles at variable time intervals prior to transcranial magnetic stimulation (TMS). Cutaneous stimulation has previously been shown to modulate the amplitude of the motor evoked potential (MEP) and to shorten the duration of the silent period (SP) evoked by TMS. In relaxed target muscles the time-dependent modulation of TMS-evoked motor responses by homotopic conditioning stimulation differed from modulation by heterotopic stimulation. Similar differences in the modulation pattern evoked by homotopic and heterotopic conditioning stimulation were observed for two distinct target muscles of the hand (abductor digiti minimi, abductor pollicis brevis muscle). Differences in modulation were maximal when the conditioning stimulation was applied 25-30 ms and 150-200 ms prior to TMS. Comparison of the modulation of the amplitudes of MEPs evoked by transcranial electrical stimulation (TES) and the modulation of those evoked by TMS suggests that differences between homotopic and heterotopic stimulation originate subcortically at 25- to 30-ms and, at least partially, cortically at 150- to 200-ms interstimulus intervals. In isometrically contracted intrinsic hand muscles the degree to which the SP was shortened reflected the location and the timing of the conditioning stimulus. Shortening was maximal when the conditioning stimulus was applied nearest to the contracted target muscle and 20 ms prior to the test stimulus. In contrast to the SP duration, the MEP size in voluntarily contracted target muscles was unaffected by the location of the conditioning stimulus. The somatotopic gradient of SP shortening was abolished when the two target muscles were simultaneously activated isometrically. Together, our findings suggest that somatotopy of input-output relationships is implemented at both a spinal and a cortical level in the human central nervous system and may also depend on the motor task involved.

Endotoxin-treated cochleas of the guinea pig were examined electrophysiologically and immunohistochemically concerning the expression of inducible nitric oxide synthase (iNOS/NOS II). One mg of bacterial lipopolysaccharide (LPS, 5 mg/ml) or mixed solution of 1 mg of LPS plus 1 mg of N(G)-nitro-L-arginine methyl ester (L-NAME, 5 mg/ml) (L-NAME/LPS) was injected into the middle ear of guinea pigs transtympanically. The electrocochleograms were measured prior to, immediately and 48 h after the injection. Immunohistological studies for iNOS followed after fixation, embedding and sectioning of the temporal bones. The threshold and amplitude of the compound action potential (CAP) became significantly worse in the LPS treated group. In contrast, the changes of the threshold and amplitude of CAP were decreased in the L-NAME/LPS group. iNOS was expressed in the stria vascularis, the spiral ligament, the organ of Corti and the spiral ganglion in the LPS group. These immunoreactivities in the L-NAME/LPS group were less intense than that in the LPS group. These results indicate that LPS has an ototoxic effect on the cochlea and that this effect could be mediated by iNOS produced high nitric oxide under inflammatory conditions.

The presence of nitric oxide synthase (NOS) in substructures of the cochlea of guinea pigs is an issue of current focus. Moreover, information concerning the localization of cells effected by the NO/cGMP-pathway are rare. Paraffin sections of guinea pig cochlea were incubated with specific antibodies to the three known NOS isoforms, soluble guanylyl cyclase (sGC) and cyclic guanosine-monophosphate (cGMP), the second messenger system of NO. While detection of inducible iNOS failed in all cochlear structures, expression of endothelial eNOS was found in the spiral ligament, in the stria vascularis, in cells of the organ of Corti, in nerve fibers and in some perikaryia of the spiral ganglion. The cochlear nerve showed an accentuated affinity for immunostaining in distal, basal segments of the cochlea. Neuronal bNOS was found predominantly in the endosteum of the modiolus and cochlea and was less intensively present in all perikaryia of the spiral ganglion and in the spiral ligament. Supporting cells of the organ of Corti and cells in the limbus spiralis displayed only modest immunostaining, while bNOS was not found in outer and inner hair cells. NOS detection was accompanied by immunoreactivity to sGC and to cGMP. The presence of NOS and its second messenger system gives evidence for a possible involvement in neurotransmission, regulation of the cochlear amplifier and in homeostasis.

Based on in vitro studies, nitric oxide (NO) is reported to be involved in initial neuronal differentiation. In order to compare this finding with the situation in vivo, we have looked for the expression of the three NO synthase isoforms in the developing mouse vestibulocochlear system. From these isoforms only the inducible NOS II is expressed during inner ear development. Examination of a series of embryonic and early postnatal animals, up to postnatal day 6, reveals a maturation-dependent, monophasic expression of this isoform. Initial expression is observed by day 10 of gestation in nerve cells of the vestibolocochlear ganglion and on their fibres. By day 14 of gestation, these afferent fibres penetrate the epithelium of the prospective receptor fields making contact with early, differentiating immunoreactive cochlear hair cells and receptor cells of the macula and crista ampullaris. This receptor-cell-derived immunoreactivity vanished in differentiated sensory hair cells by postnatal day 6, when both the constitutive isoforms and subsequent activated members of the down stream second messenger cascade (guanylate cyclase/cGMP) of the adult mouse were not then detectable. The strict phasic expression of NOS-II, independent of the second messenger system mentioned above, implies that there is a unique role for the inducible NOS isoform in nerve cell differentiation, independent of the NO/guanylate cyclase/cGMP pathway.

Since NO is believed to be involved in cochlear physiology, presence of the constitutive isoforms of nitric oxide synthase (NOS), and the target enzyme of NO, soluble guanylyl cyclase (sGC) in structures of the mammalian cochlea have been demonstrated. To date, no reports have been published regarding the detection of the inducible isoform (NOS II) in the cochlea. In order to show the capability of iNOS expression in cochlear tissue, a mixture of proinflammatory bacterial lipopolysaccharides (LPS) and tumor necrosis factor alpha (TNF-alpha) was injected into the tympanic cavity of guinea pigs, vs. saline-solution as control. Paraffin sections of LPS/TNF-alpha treated and saline-treated cochleae (6 h) were examined immunohistochemically with specific antibodies to neuronal, endothelial and inducible NOS and to sGC. Initiated expression of iNOS in the cochlea was observed in the wall of blood vessels of the spiral ligament (SL) and the modiolus, in supporting cells of the organ of Corti, in the limbus, in nerve fibers and in a part of the perikarya of the spiral ganglion after LPS/TNFalpha-treatment. iNOS was not detected in saline-treated control tissue. Expression of both constitutive NOS-isoforms (endothelial and neuronal NOS) and of sGC showed no significant differences in both experimental groups. Endothelial eNOS and neuronal bNOS were detected co-localized in ganglion cells, in nerve fibers, in cells of the SL and in supporting cells of the organ of Corti, but not in sensory cells. Strong labeling for bNOS became evident in the endosteum of the cochlea, while in the endothelium of blood vessels and in the epithelium of the limbus only eNOS could be labeled. sGC could be detected in SL, in supporting and sensory cells of the organ of Corti, in nerve fibers, ganglion cells, in the wall of blood vessels and in the limbus-epithelium. While small amounts of NO, generated by bNOS and eNOS, seem to support the cochlear blood flow and auditory function as well as neurotransmission, high amounts of iNOS-generated NO could have dysregulative and neurotoxic effects on the inner ear during bacterial and viral infections of the middle and inner ear.

Nicht immer prasentiert sich die Lyme-Borreliose bei Manifestationen im Zentralnervensystem mit ihren typischen Fruh- oder Spatinfektionssyndromen. Es wird uber eine seltene Erstmanifestation—Schizophrenie—berichtet. Das Magnetresonanztomogramm (MRT) wurde wegen Therapieresistenz initiiert und zeigte vaskulitische Lasionsmuster. Die laborchemische Analyse ergab einen erhohten Borrelientiter. Bei einer zweiten Patientin war im kranialen Computertomogramm (CT) eine Enzephalitis mit raumforderndem Charakter zu erkennen und lenkte deshalb von der Verdachtsdiagnose Lyme-Neuroborreliose ab. Ein Zeckenbis war bei beiden Patienten nicht eruierbar. Zur Klarung uncharakteristischer klinisch-neurologischer Symptome fuhren die Bildgebung, der lymphozytare Liquorbefund sowie der Nachweis borrelienspezifischer Antikorper im Serum zur Zieldiagnose Neuroborreliose.

In order to demonstrate the involvement of nitric oxide synthases (NOS)--in particular the inducible isoform (iNOS)--in inflammatory processes within the nasal airways, we used organ-bath incubation to study isolated inferior turbinates and mucosa of the maxillary sinus of guinea pigs. The pattern of the expression in various substructures of the nasal mucosa was of special interest. Mucosa was incubated for 6 h with lipopolysaccharides (LPS) produced by E. coli, interleukin II (IL-2) or tumor necrosis factor-alpha (TNF-alpha). Saline was used as the control solution. Following incubation the specimens were fixed in buffered 4% formaldehyde solution over a period of 4 h. Tissues were next exposed to nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase-reaction and immunostained with specific antibodies to iNOS. Results then showed a clearly increased or initiated expression of iNOS in epithelium, glands, leucocytes and blood vessels of treated tissues in comparison to the control specimens. The inflammatory mediator LPS and the cytokines Il-2 or TNF-alpha alone were found to be capable of increasing the expression of iNOS, although the effects of LPS clearly exceeded those of the cytokines. This finding implicates iNOS-generated nitric oxide as a key factor for causing nasal swelling, secretion and obstruction during nasal infections and allergic episodes.

Background The discrimination between atrial flutter (AFlu) and atrial fibrillation (AFib) can be made difficult by an irregular ventricular response owing to complex conduction phenomena within the atrioventricular (AV) node, known as multilevel AV block. We tested the hypothesis that a mathematical algorithm might be suitable to discriminate both arrhythmias. Objectives To discriminate AFlu with irregular ventricular response from AFib based on the sequence of R-R intervals. Methods Intracardiac recordings of 100 patients (50 patients with AFib and 50 patients with AFlu) were analyzed. On the basis of a numerical simulation of variable flutter frequencies followed by 2 levels of AV block in series, a given sequence of R-R intervals was analyzed. Results Although the ventricular response displays absolute irregularity in AFib, the sequences of R-R intervals follow certain rules in AFlu. We find that using a mathematical simulation of multilevel AV block, based on the R-R sequence of 16 ventricular beats, a stability of atrial activation could be predicted with a sensitivity of 84% and a specificity of 74%. When limiting the ventricular rate to 125 beats/min, discrimination could be performed with a sensitivity of even 89% and a specificity of 80%. In cases of AFlu, the atrial cycle length could be predicted with high accuracy. Conclusion On the basis of the electrophysiological mechanism of multilevel AV block, we developed a computer algorithm to discriminate between AFlu and Afib. This algorithm is able to predict the stability and cycle length of atrial activation for short R-R sequences with high accuracy.

Right ventricular (RV) function determines long-term outcome in many cardiopulmonary diseases. However, the assessment of RV function is time-consuming and surrogate parameters derived from two-dimensional (2D) or Doppler echocardiography show poor consistency. Forty consecutive patients were examined within 30 min after magnetic resonance imaging (MRI) with comprehensive echocardiography, including strain imaging and real-time three-dimensional echocardiography. A new parameter, the RV automated systolic index (RV-ASI), was obtained from the apical four-chamber view using semi-automated border detection. RV-ASI could be assessed by 2D echocardiography in 38 of 40 patients. RV ejection fraction assessed by MRI was 48 ± 9 %, while RV-ASI was 52 ± 11 % (r = 0.74, SEE = 6 %, p

The genetic causes of congenital heart diseases especially cardiac valve disorders are mostly unknown. During the last decade, the zebrafish became an excellent and established model organism (1) to uncover these genetic defects and (2) to elucidate the underlying molecular pathomechanisms. We recently isolated the zebrafish mutation ping pong ( png m683 ) in a large-scale ENU-mutagenesis screen for recessive lethal mutations that perturb cardiac function. png mutant zebrafish embryos show pathologically developed cardiac valves. Due to malformation of the cardiac AV valves, png mutant zebrafish embryos exhibit vigorous regurgitation of blood between the atrium and the ventricle. Furthermore, as a result of the cardiac valve malformation and cardiac dysfunction png mutants die at day 6 post fertilization. Expression of several factors known to be crucial for the proper development and formation of the atrio-ventricluar canal (e.g. notch1b, bmp4 or versican) is significantly altered in png mutant zebrafish hearts. By a positional cloning approach we demonstrate that the ping pong phenotype is caused by a promotor mutation in a zebrafish gene encoding for a novel component of the “transcriptional mediator complex”. This mediator complex is a multi-protein complex that acts as a transcriptional coactivator and transduces informations from transcription factors to the RNA polymerase II. png is strongly expressed in zebrafish as well as human cardiomyocytes. Furthermore, sequence alignments demonstrate the evolutionary conservation of the ping pong gene product. Gene specific knock-down studies by means of modified antisense oligonucleotides reveal a phenocopy of the png mutant phenotype whereas injection of the gene-specific mRNA in png mutant embryos restores the mutant phenotype indicating that png is indeed responsible for the observed phenotype. The zebrafish evolved as an excellent model organism to study the molecular signalling pathways involved in cardiac valve formation. By detailed characterization of the zebrafish line ping pong we will obtain new insights into these molecular mechanisms especially the transcriptional control of valve formation and therefore the pathomechanisms of human cardiac valve disorders.

The protein kinase Akt (protein kinase B) can be activated by numerous growth factors via PI-3 kinase-generated phosphoinositides and is thought to have anti-apoptotic properties. Activated Akt/PKB boosts the activity of endothelial NO synthase (NOS III), which has been found in the key areas of the inner ear (e.g., hair cells and stria vascularis). In order to localize activated Akt/PKB (phospho-Akt) in the cochlea of guinea pigs, sections of ten temporal bones were observed immunohistochemically. The strongest immunoreactivity was found in and underneath inner hair cells (IHC). Within the organ of Corti, reactivity was found in supporting cells, while outer hair cells remained unstained. Spiral ganglion cells, the endothelium of the lateral wall and the vascular area of the modiolus showed moderate staining. The results give evidence that activated Akt/PKB influences the activity of the NO/cGMP pathway in the cochlea. Because of the antiapoptotic properties, activated Akt should now be examined under non-physiological conditions.

The antioxidant protein peroxiredoxin (Prx) I is a thioredoxin peroxidase that is involved in the regulation of proliferation and differentiation of mammalian cells. Here, it is shown that Prx I gene expression was induced transcriptionally by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in cultured rat liver tissue macrophages and RAW264.7 monocytic cells. TPA-dependent induction of Prx I gene expression was mediated by two proximal activator protein-1 sites of the rat Prx I promoter region that were nuclear targets of c-Jun as determined by transfection studies with luciferase reporter gene constructs and electrophoretic mobility shift assays. The transcription factor Nrf2, however, was not involved in the regulation of Prx I promoter activity. Prx I gene induction by TPA was decreased by protein kinase C inhibitors and overexpressed dominant negative forms of Ras and MEKK1, but not Raf-1. The p38 MAPK inhibitor SB202190 and overexpression of dominant negative mutants of MAPK kinase 4 (MKK4), MKK6, and p38 inhibited the TPA-dependent induction of Prx I gene transcription. In contrast, inhibitors of the JNK, SP600125, and the NF-kappaB signaling pathway, caffeic acid phenethyl ester, respectively, as well as overexpressed dominant negative MKK7 and IkappaB, had no effect on the up-regulation of Prx I reporter gene activity by TPA. Cotransfection of wild-type p38alpha and p38beta, but not that of p38gamma and p38delta, increased Prx I promoter activity. The data indicate that a protein kinase C, Ras, MEKK1, p38 MAPK signaling pathway plays a major role for the transcriptional up-regulation of Prx I gene expression.

Cisplatin is reported to damage the stria vascularis of the cochlea. Free radicals, especially large amounts of nitric oxide catalyzed by inducible nitric oxide synthase, are considered to have an important role in this toxicity. The induction of inducible nitric oxide synthase is regulated by nuclear-factor kappa B (NF-kappa B). We examined the damage of the stria vascularis by immunohistochemical techniques.Cisplatin (15 mg/kg b.w.) was injected intraperitoneally into the mice. Three days after the injection, the cochleas were immunohistochemically-stained using specific antibodies for nuclear-factor kappa B (NF-kappa B), inducible nitric oxide synthase (iNOS) or single-stranded DNA.NF-kappa B was expressed in the cisplatin-treated cochlea, especially in the stria vascularis and the spiral ligament. iNOS was also expressed in the stria vascularis and the spiral ligament. Fragments of DNA were observed only in the stria vascularis.The large amounts of NO catalyzed by iNOS led to inner ear dysfunction. Our results indicate that apoptosis is triggered by iNOS and that it mediates the ototoxicity induced by cisplatin.

Growth factors such as vascular endothelial growth factor (VEGF) exert their proliferative properties partly through activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2). Although both VEGF and inactive ERK could be detected in the inner ear of guinea pigs, under normal conditions activated ERK (phospho-ERK) was found only sparely. Cochleae of adult guinea pigs were removed, incubated with VEGF in a carbogen-gased organ-bath for 5, 15, 30 and 60 min (n=6 in each group), fixed with PFA 4%, embedded in paraffin and sectioned, followed by immunohistochemical staining to inactive and active ERK. Whereas inactive ERK was found in all cochleae, in sensory and supporting cells of the apex activated ERK was strongly detected after 5-min VEGF-incubation. After 15 min all Corti-organs showed clear staining corresponding to activated ERK, which decreased again after 30 min. Faint staining in endothelial cells of the spring-coil-vessels and in the spiral ganglion cells was found after 30 min and was increased after 60 min, while the staining in the Corti-organs vanished. Addition of the MEK-inhibitor PD 98059 to the organ-bath led to diminished phospho-ERK1/2 immunostaining. These findings provide evidence for a VEGF-dependent phosphorylation of ERK1/2 in the cochlea. Activated ERK1/2 is thought to support axonal outgrowth, enhancement of cell survival and to regulate the turnover of the NO/cGMP-pathway.

Background: Cisplatin (CDDP) is known to cause inner ear damage while carboplatin (CBDCA) induces less ototoxicity than CDDP. We examined apoptotic changes in the cochlea of guinea pigs after injection of CDDP or CBDCA using immunohistochemical and electrophysiological techniques. Methods: Three days after the injection of each solution, the cochleas were immunohistochemically examined for the presence of fragments of single-stranded DNA (ssDNA). The auditory brain stem response was recorded before and three days after the injection. Results: We detected fragments of ssDNA in the stria vascularis and the spiral ligament of the CDDP-treated cochlea. In this group, the threshold of the auditory brainstem response was significantly elevated, however, in the CBDCA group, no apparent change of the threshold was detected. In the CBDCA group, fragments of ssDNA were detected in the stria vascularis and the spiral ligament. The number of cells that stained positive for ssDNA, was less than that in the CDDP group. Conclusions: Our findings indicate that CBDCA induces less apoptosis than CDDP and that this phenomenon contributes to the ototoxicity of CDDP.

This study was undertaken to examine, electrophysiologically and immunohistochemically, the effect of endotoxin on the guinea pig cochlea. A bacterial endotoxin (lipopolysaccharide, LPS, 5 mg/ml, 0.2 ml) was injected into the middle ear trans-tympanically. The electrocochleograms were continuously recorded from before to 48 h after the injection with an electrode inserted into the facial canal. Then, the animals were sacrificed by intracardiac perfusion of a fixative, temporal bones were removed and immunohistochemically stained for single-stranded DNA (ssDNA) and caspase 3 (CPP32). ssDNA was detected at 48 h in the stria vascularis and spiral ligament. CPP32 was observed in the stria vascularis, the spiral ligament and the organ of Corti. The threshold of the compound action potential increased significantly at 48 h in the LPS group. These results suggest that the activation of CPP32 and fragmentation of DNA are involved in the dysfunction of the cochlea observed under inflammatory conditions.

A 37-year-old male patient presented to the stroke unit of our neurology department with weakness and paresthesia of the right arm and slight anomia. Physical examination revealed distal palsy of both arms, predominantly on the right side. No other pathologic findings were observed. Cranial MRI revealed bilateral territorial infarction in the supply territories of the middle and posterior cerebral arteries. A 12-lead electrocardiography showed incomplete right bundle branch block, negative T waves and ST depression in II, III, aVF and V4–V6 (Fig. 1a). Transesophageal and transthoracic echocardiography revealed normal dimensions of both ventricles with regular systolic and diastolic function. No intracardiac thrombi and no patent foramen ovale could be detected at that time. HolterECG did not show paroxysmal atrial fibrillation. Carotid artery stenosis was ruled out by duplex ultrasound. Blood examination showed increased creatinine (1.59 mg/dL) and urea (60 mg/dL). The white cell count was 13.2 9 10/L, with 6.9 9 10/L eosinophils (52%). A screening for vasculitis was unremarkable aside from an ANA titer of 1:320. Stool analysis and serologic tests were unsuspicious for parasitic helminth infection and there was no apparent primary allergic disorder. Because of the drastically increased eosinophils, we suspected a myeloproliferative disorder. A bone marrow biopsy was done, showing increased myelopoiesis and megakaryopoiesis as well as massive eosinophilia. These findings led us to the diagnosis of hypereosinophilic syndrome (HES). The firstline therapy for HES is steroids [3]; so, we started treatment with prednisone. Antiplatelet therapy with aspirin was administered because of the stroke. As it may have influence on the treatment, it is necessary to further classify the disease by cytogenetic and molecular genetic analysis. During these diagnostic procedures, a chromosomal deletion on 4q12 leading to creation of the FIP1L1–PDGFRA fusion gene was detected. This gene encodes a protein displaying constitutive tyrosine kinase activity. Thus, the final diagnosis was F/P ? chronic eosinophilic leukemia (CEL) [7] and the tyrosine kinase inhibitor imatinib, which is regarded as first line therapy for this special disease subgroup [3, 7], was administered at a dose of 100 mg/day. A control echocardiography was performed 2 months later, revealing an elastic echogenic mass filling the left ventricular apex (Fig. 1b; Movie 1) and right ventricular involvement. These findings could be confirmed in further follow-up studies. Cardiac magnetic resonance (CMR) also confirmed the presence of thrombotic formations in the left and right ventricular apex (Fig. 1d). Strikingly, leukocyte count was normalized to 9.1 9 10/L with 0.5% eosinophils at this point of time. No FIP1L1–PDGFRA transcripts were detectable, so a complete molecular genetic remission was achieved. The patient remained clinically stable without symptoms. Echocardiography follow-up was continued and after 1 year of therapy with imatinib, a clear regression of cardiac involvement could be assessed, since the left and right ventricular echogenic masses could not be detected any longer (Fig. 1c; Movie 2). Chronic eosinophilic leukemia is part of the heterogenous group of HESs. As these are very rare, there are no published Electronic supplementary material The online version of this article (doi:10.1007/s00392-010-0143-9) contains supplementary material, which is available to authorized users.

Cisplatin is known to cause inner ear damage (ototoxicity). The role of inducible nitric oxide synthase (iNOS) in the cochlea of guinea pigs after injections of cisplatin or a combination of cisplatin and NOS inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) i.p. was examined electro- and immunohistochemically. The auditory brain stem responses (ABR) were measured prior to injection and 3 days after the injection. Three days after injection, the cochleas were examined immunohistochemically for iNOS. We found that iNOS was expressed in the cisplatin- and L-NAME/cisplatin-treated cochlea. The threshold shift of ABR was significant in the cisplatin group, whereas it was decreased in the L-NAME/cisplatin group. iNOS catalyzed high NO levels lead to inner ear dysfunction. Our results indicate that iNOS mediates the ototoxicity of cisplatin.