Hearing deficits associated with cognitive impairment have attracted much recent interest, motivated by emerging evidence that impaired hearing is a risk factor for cognitive decline. However, dementia and hearing impairment present immense challenges in their own right, and their intersection in the auditory brain remains poorly understood and difficult to assess. Here, we outline a clinically oriented, symptom-based approach to the assessment of hearing in dementias, informed by recent progress in the clinical auditory neuroscience of these diseases. We consider the significance and interpretation of hearing loss and symptoms that point to a disorder of auditory cognition in patients with dementia. We identify key auditory characteristics of some important dementias and conclude with a bedside approach to assessing and managing auditory dysfunction in dementia. Although hearing impairment is not generally regarded as a cardinal feature of dementia, hearing in patients with dementia is a focus of growing clinical interest. Recent evidence suggests that hearing loss may predict or accelerate cognitive deterioration –3, and aIn this review, we outline a clinically oriented, symptom-based approach to hearing in dementia, informed by recent progress in the clinical auditory neuroscience of these diseases. We consider the problem of hearing loss (impaired detection of sound and how this interacts with cognitive function) and symptoms that point to a disorder of auditory cognition . We identify key auditory characteristics of some important dementias. We conclude with a bedside approach to assessing and managing auditory dysfunction in dementia.Hearing  has been aligned with other complex neuropsychological processes based on studies of the normal brain and focal brain damage , 13. TogAs a framework for analysing disorders of auditory cognition, it is useful to consider complex sounds  as ‘auditory objects’ that must be disambiguated from the auditory background and organised into coherent perceptual representations . The proDementia is arguably the most significant public health problem confronting ageing societies, with an estimated 800,000 sufferers currently in the United Kingdom alone . HoweverSignificant hearing loss  affects around 40 % of those aged over 65  and has While the association between hearing loss and cognitive decline appears robust, the mechanism remains unresolved. Hearing impairment might accelerate cognitive decline by compounding sensory and social isolation, increasing cognitive load, and thereby exhausting compensatory cognitive reallocation, or constitute a nonspecific marker of frailty . HoweverAuditory deficits in AD may be disproportionate to any abnormality of sound detection or otological markers , 30–34: The effects of hearing impairment on cognitive decline might be most parsimoniously considered as an interaction of peripheral and more central factors. The auditory system has extensive efferent as well as afferent traffic  allowingSyndromic associations of dementia with dysfunction of cochlea or ascending auditory pathways are uncommon and generally occur in the context of more complex neurological impairment, often in younger patients; examples are summarised in Table Though auditory dysfunction is rarely the presenting feature, histopathological involvement of auditory cortices has been described in major neurodegenerative dementias , 38–41, Here, we emphasise the differential diagnosis of auditory symptoms; characterisation of auditory deficits using neuropsychological tests is a complementary enterprise. Together, these approaches define auditory phenotypes, summarised for selected dementias in Table Patients with dementia may have reduced perception of sound disproportionate to any damage involving cochlea or ascending auditory pathways: this may manifest as cortical deafness  or Frank auditory disorientation is uncommon in dementia though does occur  in patients with posterior cortical degenerations . HoweverOther symptoms, experienced particularly by patients with AD and progressive nonfluent aphasia suggest auditory apperceptive dysfunction: disproportionate difficulty identifying or understanding sound objects under unusual or degraded listening conditions. The patient may not recognise a familiar voice over a noisy telephone line or may fail to understand a message delivered in an unfamiliar accent , 52). SuPatients with semantic dementia develop deficits of nonverbal sound recognition (auditory associative agnosia) as part of a pan-modal erosion of semantic memory, linked to antero-mesial temporal lobe dysfunction , 53, 54.Patients with semantic dementia commonly report tinnitus , linked to structural alterations in a fronto–temporo–subcortical network ; while hAltered emotional or ‘hedonic’ behavioural responses to sound are increasingly recognised in patients with dementia. Impaired processing of emotional prosody has been described in AD, Parkinson’s disease, behavioural variant frontotemporal dementia, and progressive aphasia syndromes –65, and A clinical framework for assessing and managing the patient presenting with cognitive impairment and altered hearing is outlined in Table Hearing function should be assessed in all patients receiving a diagnosis of dementia: to identify a factor that may be detracting from quality of life and impeding care , to gaugIn patients with known hearing impairment, any supervening cognitive decline should be thoroughly characterised. This is particularly challenging in those with pre-lingual profound hearing loss. British Sign Language (BSL) users form a close-knit minority group defined culturally as well as linguistically; they often have difficulty in accessing appropriate assessment services and, therefore, may have more advanced dementia when diagnosed. Ideally, assessment of such individuals should be undertaken in a clinic with special expertise in working with deaf people, engaging a neuropsychologist with relevant communication skills and expertise and BSL interpreters trained in the requirements of cognitive assessment. Until recently, there were no normative data on cognition in deaf subjects; however, a cognitive screening instrument based on British Sign Language has now been developed . As withPure tone audiometry and otoacoustic emissions  are relatively simple and well-tolerated techniques to assess cochlear and ascending auditory pathway function in cognitively impaired patients. Measurement of speech-in-noise perception Table  more cloWhile effects on specific aspects of cognitive function are difficult to predict, correction of reversible hearing deficits has been shown to benefit global functioning in daily life , 78, 79.Hearing has long been the poor relation of memory and vision in the cognitive clinic. The emerging epidemiological evidence may shortly transform this situation. The comprehensive assessment of hearing in dementia presents both challenges and opportunities. There is a need to develop practical and reliable tests that can disambiguate the effects of peripheral hearing and auditory cognitive dysfunction, to develop auditory interventions directed to cognitively impaired people and to assess these systematically and longitudinally in a range of dementias, referenced to healthy older people. In addition to capturing disability and improving quality of life, a more detailed picture of the spectrum of auditory dysfunction in dementia would have considerable neurobiological and clinical resonance. Sound is a dynamic and computationally demanding sensory signal that engages complex emotional and other behaviours: the processing of sounds taxes brain networks targeted by neurodegenerative pathologies and may yet yield novel cognitive ‘stress tests’ for diagnosis and treatment tracking in these diseases.


Exostosis in external auditory canal is common among surfers. Common symptoms are decreased hearing or loss of hearing, ear infection, and/or plugging sensation. Repeated exposure to cold water is a key clinical history to suspect this condition. Based on symptoms and existence of infection, surgical removal of the exostosis is recommended. An otherwise healthy 46‐year‐old man presented with hearing disturbance in his right ear of 1 day duration, after having gone surfing. Otoscope showed narrow external auditory meatus due to protruded mass Fig. . There wDetailed history is important to help diagnose surfer's ear when patient is complaining of hearing disturbance.YO: drafted the article. All authors participated in critical review and the revision of the articles. All authors gave the final approval of the article. All authors have accountability for all aspects of the work.None declared.


A 45-year-old man presented with a thunderclap headache. Brain computed tomography (CT) scan without contrast showed massive subarachnoid hemorrhage (SAH). We transferred him to the angiography unit where he was diagnosed with a left (Lt) distal internal carotid artery (ICA) aneurysm. We secured the aneurysm using 8 coils. He was stable the following days. 1He was monitored via daily transcranial sonography (TCS) for the potential development of vasospasm. In TCS, the coiled distal ICA aneurysm was visualized as a round hyperechoic mass anterior to the midbrain. There was no aneurysm refilling on consequent evaluations. Unenhanced TCS is proposed as a screening tool for coiled aneurysm refilling.


A 59-year-old female presented to the emergency department (ED) three days after accidental ingestion of an intact in-the-ear hearing aid. This is the first report of ingestion of a complete hearing aid traveling past the gastroesophageal junction. Of concern was the exposed battery attached to the hearing aid that had advanced minimally in the three days since last evaluation. This case report discusses her ED testing, including gastroenterology consultation, and ultimately retrieval from her distal stomach. The authors conclude that this removal was not medically necessary. Cochlear implants enhance sound transmission to the vestibulocochlear organ, and miniaturized within-ear devices amplify and compensate for conductive and sensorineural hearing loss.Hearing loss occurs in some 30% of adults greater than 60, and has been associated with increased risk of dementia and falls.In-the ear (ITE), in-the-canal, and completely-in-canal hearing aids are devices small enough to cause risk of ingestion in the elderly or cognitively impaired. Foreign body ingestion is a relatively common chief complaint in the emergency department (ED), but most cases occur in children with peak incidence between six months and six years of age. Eighty percent of foreign bodies pass spontaneously through the gastrointestinal tract; surgical intervention is required in only 12–16%. Death is extremely rare. One study reported no deaths among 852 adults, while another in children reported one out of 2,206.We present the first reported case of an adult accidently ingesting a complete hearing aid with exposed battery.A 59-year-old Caucasian female presented to the ED with a chief complaint of accidentally swallowing her hearing aid. The patient denied symptoms aside from anxious thoughts regarding the ingestion. She swallowed the device accidentally three days before, when she grabbed a handful of acetaminophen from her night stand, which she takes each morning for osteoarthritis. The patient went to work on the day of the ingestion, but left early given mild discomfort in her chest and concern about the swallowed hearing aid. An abdominal radiograph done at an outside hospital revealed the foreign body at the gastroesophageal (GE) junction. The patient was sent home and instructed to carefully examine her stool for passage of the foreign body.On presentation to our ED, the patient’s chest discomfort was resolved; however, she was concerned because she had not seen the foreign body in any of her stools. She had taken polyethylene glycol 3350  daily in hopes this would promote passage. She denied shortness of breath, cough, dysphagia or odynophagia, abdominal pain, nausea, vomiting, or changes in bowel habits. The patient brought her matching hearing aid, which was not ingested: an ITE device, approximately 2cm in diameter with plastic casing. However, when the hearing aid is in the off position, as it was when it was ingested, a small battery protrudes from the device and is not protected within plastic casing .The patient had osteoarthritis, depression, and attention deficit hyperactivity disorder, with two caesarean sections, and she denied tobacco, alcohol, or illicit drugs.2 saturation 100% on room air. Tachycardia was resolved by the time of exam by the physician. Oropharynx revealed moist oral mucosa, no pharyngeal erythema, exudate or fullness, and uvula was midline. There was no reproducible chest wall discomfort or subcutaneous crepitus of the chest. Breathing was non-labored with no accessory muscle use. Breath sounds were clear and equal bilaterally with no wheezes or rhonchi. Cardiac auscultation was normal. Radial pulses were normal and equal. Her abdomen was soft, non-distended, and non-tender, with normal bowel sounds.On physical exam, the patient was well-appearing, in no apparent distress and was breathing comfortably. Initial vital signs were temperature 36.6C, blood pressure 153/93 mmHg, heart rate 106 beats per minute, respiratory rate 16 breaths per minute, and OAn abdominal radiograph was done to localize the foreign body, as the patient reported the hearing aid was easily identifiable in the distal esophagus on plain films three days prior .Final read of the plain films was: “3 closely grouped metallic densities measuring 2 mm, 6 mm and 12 mm in size project over the midline upper abdomen at the level of L1–2. Findings may represent the hearing aid/foreign body of interest.”Emergency physicians (EP) consulted the gastroenterology service after the radiographs were shot; however, neither team could determine the precise location of the foreign body from these films alone. Non-contrast computed tomography (CT) of the abdomen and pelvis was then obtained per GI’s request for further evaluation of the foreign body’s size and location  and 4.While awaiting CT results, EPs spoke with the American Association of Poison Control Centers (1-800-222-1222) regarding this case. Their representative reassured us that exposed batteries are only a concern if they are still in the esophagus, due to constant contact with the esophageal mucosa, which allows for injury of the mucosa. Assuming the hearing aid was at least beyond the esophagus by that point, the patient was expected to safely pass the foreign body in the coming days.The final radiology report concluded that the foreign body was in the mid-upper abdomen, but could not confidently state the exact location.The GI team determined the object appeared to be in the distal gastric body/antrum. After examining the patient’s other hearing aid, and the size of the object on CT, they felt the object would eventually pass uneventfully, even if the exposed battery were to entirely separate from the hearing aid. However, as the patient continued to express concern, she was offered an esophagogastroduodenoscopy.The patient had a successful endoscopy under general anesthesia the following morning with retrieval. The hearing aid was removed intact with battery still in place using a Roth net. There was no evidence of esophagitis, erosions, or ulcerations. The patient tolerated the endoscopy well with only a mild sore throat.This is the first report of ingestion of an ITE hearing aid traveling past the gastroesophageal junction. We could find only one other case of an 86-year-old man accidentally ingesting a whole hearing aid, but this was a larger behind-the-ear (BTE) device.While foreign body ingestions are common, this case is unique because a potentially dangerous exposed battery remained in the stomach for three days with minimal advancement. Exposure of stomach mucosa to the battery with potential for impaction proximal to the pylorus is a unique situation. Consensus between EPs, GI and poison center determined the situation to be non-emergent. Although comforting to the patient, we believe that endoscopic removal of the hearing aid, despite the exposed battery, was unnecessary and it would have passed given sufficient time.


To relate the performance of individuals with hearing loss at high frequencies in speech perception with the quality of life before and after the fitting of an open-fit hearing aid (HA). The WHOQOL-BREF had been used before the fitting and 90 days after the use of HA. The Hearing in Noise Test (HINT) had been conducted in two phases: (1) at the time of fitting without an HA (situation A) and with an HA (situation B); (2) with an HA 90 days after fitting (situation C). Thirty subjects with sensorineural hearing loss at high frequencies. By using an analysis of variance and the Tukey’s test comparing the three HINT situations in quiet and noisy environments, an improvement has been observed after the HA fitting. The results of the WHOQOL-BREF have showed an improvement in the quality of life after the HA fitting (paired t-test). The relationship between speech perception and quality of life before the HA fitting indicated a significant relationship between speech recognition in noisy environments and in the domain of social relations after the HA fitting (Pearson’s correlation coefficient). The auditory stimulation has improved speech perception and the quality of life of individuals. Hearing aid users show difficulty in perceiving speech when in the presence of competing background noise, which may affect their social interaction. The auditory deprivation causes consequences in the individual’s life and affects the ability to properly understand acoustic information, as well as in the way they relate to their environment, which can cause a decisive impact on their quality of life (QoL).Individuals who exhibit hearing loss restricted to high frequencies (above 1 kHz) in adverse conditions, such as when speech is distorted, or in the presence of noise, may face several difficulties in speech intelligibility because the number of auditory cues drops considerably. The speech intelligibility depends on the consonant sounds that present sound spectrum with frequencies above 2 kHz. The fact that the consonants are low intensity sounds in relation to vowels makes them more difficult to detect, especially for individuals with slope hearing lossThe OF is a specific miniature behind the ear (BTE - HA) that uses a thin sound tube and a soft vented silicone eartip holding the tube in place inside the canal without using an earmold. Open-fit does not only refer to the HA model, but also to the EAC condition not being occluded, which can be verified through measurements with probe microphone real-ear occluded response (REOR) equal to the real-ear unaided response (REUR) allowing the use of the term “open-fit” throughout the article.One way to measure success in the HAs fitting is to assess the improvement in speech recognition, particularly in noisy environments, which could possibly lead to an improvement in the individual QoL.,,,,-Over the years, many tests of speech perception in noise have been developed in an attempt to better evaluate the individual’s performance in noisy environments, seeking to maximize the approach in situations of daily life such as CTS - Connected Speech Test, SIN - Speech in Noise Test, QuickSIN-QuickSpeech-in-Noise Test, BKB-SIN - Bamford-Kowal-Bench Speech-in-Noise Test, SPIN - Speech Perception In Noise, and HINT – Hearing in Noise Test. There are differences between the tests in the speech materials that influence the assessment of speech intelligibility in noisy environmentsLiterature shows that the verification and validation in the fitting of HAs decreased the number of returns and increased the patient’s satisfaction with HAs. Thus, the author suggested perception of speech in noise tests for validationSeveral studies point to a negative assessment of QoL in individuals with hearing loss, but few studies have used specific instruments to measure the QoL, such as the WHOQOL-Breef, related to hearing loss. These surveys are scarce in the literature, which makes this topic a necessary study subject in the audiology areaSlope hearing loss configurations are prevalent in audiological clinics. Subjects with the slope hearing loss do not often feel hearing difficulties in the silence, thus they do not present hearing complaints, since they have normal hearing up to 1 kHz and moderate loss to high tones, which causes difficulty only in noisy environments. The indication of HA for this population should be based on the difficulty presented in noise. The speech perception test is an important tool that can objectively show the improvement for the patients, therefore improving their quality of life.Studies on the QoL and the speech perception of individuals with hearing loss at high frequencies may fundamentally contribute to the field of audiology, aiding and/or confirming the decision-making process regarding the prescription of HAs and providing more support for the counseling and guidance processes in the use of OF fitting.The aim of this study was to analyze the relationship between speech perception and QoL in adults and elderly with hearing loss at high frequencies, before and after the adaptation of OF HAs.This prospective cohort study has been conducted after approval by the Ethics Committee for Research in Humans (607.178) and after the agreement and informed consent of the subjects.The study included 30 individuals who have met the following inclusion criteria:Aged more than 30 years;Diagnosed with sensorineural hearing loss with a slope hearing loss, restricted at high frequencies (above 1 kHz), compatible with a conventional OF HA (Receiver in the advice - RITA) fitting, and with digital technology;No prior experience with the use of HAs;Absence of other pathology associated with hearing loss;Ability to understand the WHOQOL-BREF questionnaire. If subjects had any difficulties in answering the questions, they would be excluded from the sample.The sample consisted of 11 female and 19 male aging from 34 to 78 years . The audiometric threshold averages are in In order to select the type and model of HA, the audiological features and communicative needs of the participants have been analyzed. According to this analysis, the OF HAs were selected. The HAs selected was the miniature digital BTE type, with four channels, a directional microphone system, digital noise reduction, and feedback cancellation by reversed phase technology. All HAs were the same manufacturer and model.The HAs have been programmed via HI-Pro and the participant’s identification data, such as name, date of birth, gender, and audiometric thresholds, have been input into the NOAH platform version 3.0 .When programming the enterprise software, the level of user experience has been defined as “experienced”, with the aim to reach target. Information related to the acoustic characteristics of the thin tube and probes have been added. The NAL-NL1 prescriptive method has been used based on the previously entered thresholds. The algorithms for digital noise reduction and directional microphones have been activated for all participants.After programming the HAs, a verification procedure using measurements from a probe microphone has been performed. This procedure has been performed in an acoustically treated room using Affinity 2.0 equipment . The following measurements have then been performed: real-ear unaided response (REUR), open-fit calibration, real-ear occluded response (REOR) always equal to the real-ear unaided response (REUR), and real-ear aided response (REAR).The values of the REAR at frequencies from 0.25 to 6 kHz for input levels of 50, 65, and 80 dB SPL have been respectively compared with the NAL-NL1 targets. Responses were considered equivalent when the difference between the target and the REAR value did not exceed 10 dBThe assessment of speech perception has been performed using the HINT adapted to Brazilian PortugueseSentences presented in a quiet environment (quiet);Sentences presented in competitive noise; the type used was masking composite noise, with only the frontal position noise being used (noise front);In both conditions the sentences have been presented first unaided and then aided.The HINT test have been performed in three situations and divided into two phases as follows:Phase 1: at the time of HA fitting in situations unaided (situation A) and aided (situation B).Phase 2: aided 90 days after the HA fitting (situation C).The test has been conducted in an acoustically treated room, using free-field sentences. In all evaluation conditions, the signal (speech and noise) was presented from a single speaker at 0° azimuth, 1 m from the listener at a height of the head. The system calibration has been performed by placing a microphone at the reference corresponding to the participant’s head center location and 1 m away from the speaker.A list of 20 sentences has been randomly presented by the HINT PRO software for each condition. Participants have been instructed orally as to the guidelines contained in the HINT manual.The sentence was considered correct by the measurer when all essential words were repeated correctly. In this case, the examiner pressed the “yes” button on the software screen, and the next sentence was presented at 2 dB below the intensity of the previous sentence.For the sentences in quiet condition, the presentation level was initially set at 45 dB(A). The presentation level was increased in steps of 2 dB until the participant correctly repeated the sentence. The score for this test was expressed as dB(A) level, at which the participant correctly repeated 50% of the sentences.The level of noise was fixed at 65 dB(A) for the sentences and the presentation level was initially set at 65 dB(A). The level of presentation of the sentences varied in the same manner as described for the sentences in quiet condition. The score was expressed in dB as being the SNR after the presentation of a list of 20 sentences. Therefore, the lower the SNR is, the better the speech perception for the participant under this condition.The WHOQOL-BREF questionnaire was used to assess different aspects of QoL of the participants. This questionnaire is an abbreviated version of the WHOQOL-100 developed by the World Health Organization and validated in Brazil by Fleck, et al.The WHOQOL-BREF consists of 26 questions with two pertaining to general QoL issues and the other 24 questions representing each of the 24 facets that compose the original instrument (WHOQOL-100). Thus, the 24 questions cover four domains  and each facet is represented by a questionPhysical domain: corresponding to issues related to pain, discomfort, energy, fatigue, sleep, resting, mobility, activities of daily living, dependence on medication or treatment, and the ability to work.Psychological domain: corresponding to questions about positive feelings, thinking, learning, memory and concentration, self-esteem, body image and appearance, negative feelings, spirituality, religion, and personal beliefs.Environment domain: corresponding to questions about physical safety and protection, home environment, finances, health care, social care, accessibility and quality of opportunities to acquire information and skills, opportunities and participation in recreation and leisure, the physical environment (pollution/noise/traffic/climate), and transportation.Social relations domain: corresponding to questions about personal relationships, social support, and sexual activity.The WHOQOL-BREF includes four types of response scales: intensity (ranging from nothing to intense); capacity (ranging from none to full); frequency ; and evaluation (ranging from very dissatisfied to very satisfied and very bad to very good), all being graded in five levels. The questions are scored from 1 to 5, and the scores are reversed for questions 3, 4, and 26, in such a way that 1=5; 2=4; 3=3; 4=2; and 5=1.After the questionnaire had been completed, general and domain  values were calculated, allowing for an evaluation of the individual QoL. This analysis has been performed according to the syntax described by the translators using the Statistical Package for Social Science (SPSS) 10.0 for Windows.The participants completed the questionnaire before the fitting of the HAs and after 90 days of the fitting.The results have been analyzed using descriptive and inductive statistical analysis. All statistical procedures have been performed on STATISTICA software version 5.1 . The level of significance had been set at 5% in all cases.In the comparison of speech perception (HINT) at the time of fitting in situations without an HA, with HAs, and with an HA post- fitting, analysis of variance and Tukey’s test have been applied. Comparisons of the results of the WHOQOL-BREF unaided and aided have been performed using paired t-tests. The Pearson’s correlation test has been applied to determine whether there has been a correlation between speech perception and QoL scores in the different domains of the WHOQOL-BREF.The results of the HINT at the time of fitting in situations unaided (situation A), aided (situation B), and aided 90 days after HA fitting (situation C) are shown in The analysis of variance and Tukey’s test comparing the three HINT situations under quiet and noisy conditions have showed significant improvements comparing situation A to B (0.00012) and A to C (0.00076), but there has been no difference between situations B and C (0.17659).The results of the WHOQOL-BREF unaided and aided are described in Comparisons of the results of the WHOQOL-BREF in situations A and C have showed significant improvements in all domains by paired t-tests (p>0.005), with the largest difference in the social relations domain, followed by the self-assessment of QoL domain.The relationship between speech perception and QoL in situations A and C has been performed using Pearson’s correlation coefficient, and the values of p and r are described in The HINT has been used as an outcome measurement for HAs. The mean threshold for speech recognition in quiet conditions aided (situation B) was 5.31 dB lower than unaided (situation A). Comparing the results after 90 days of fitting to the results without an HA (situation C and A), the difference was 3.74 dB. These differences were statistically significant  and demonstrated that amplification provides better speech perception in quiet,In relation to the performance in speech perception in noise, the SNR scores comparing first situations A and B, then situations A and C were respectively 0.5 dB and 2.11 dB lower. These differences were also significant . It is important to note that at the time of this evaluation, the algorithm of directional microphones was enabled, which may have contributed to an improved speech perception in noisy environments, since some studies have indicated the advantages of using this algorithms for improved speech perception in noisy environmentsThese findings are extremely significant, especially when comparing the performance of OF HAs with directional microphones and OF HAs to omnidirectional microphones. The literature shows that speech recognition performance in noisy environments using OF devices with omnidirectional microphones resembles the performance unaided. In order to ensure that this variant of the HA has a benefit, it is essential to use a directional microphoneDifferences between HINT scores unaided and aided have been clear (comparing situations A to B and A to C), but the results immediately after the fitting and those obtained after 90 days (situation B to C) were very similar for silence. These differences were 1.67 dB and 0.59 dB of noise. Considering the standard deviation for the Portuguese HINT,The evaluation of QoL has shown significant improvements in all domains after HA fitting. An improvement in social relations was expected, since individuals who are able to listen better are consequently better socializers, participate more actively in groups, and avoid social isolation caused by hearing lossSome researches demonstrated improvement only in the psychological domain. These findings could be explained as a result of the short duration of use of HAs, since the re-evaluation questionnaire has been conducted just 1 month after the fitting, and the individual might have been in the acclimatization process during this periodData from this study demonstrate that the smallest change after HA fitting occurred in the physical domain. This can be justified by the mean age of the subjects evaluated (61.96 years) involving issues related to pain, discomfort, energy, fatigue, sleep, resting, mobility, activities of daily life, dependence on medication or treatment, and ability to work,In the literature, few papers have related QoL with hearing loss using the WHOQOL-BREF. Some studies have investigated the relationship among QoL, age, sex, and the presence of hearing impairment, and found that these factors were not associated with hearing impairmentWhen we compared the results between the HINT and WHOQOL-BREF in situation A, there was no significant relationship. This can be justified by the fact that the individuals selected for this study showed a slope hearing loss with hearing preserved to 1 kHz, and in most cases, individuals with this audiometric configuration demonstrate good speech recognition performance in quiet situationsComparing the results between the HINT and WHOQOL-BREF in situation C, a significant relationship, only between HINT noise and the domain of social relations, has been observed. These results are significant because they demonstrate that individuals with better speech perception in noisy environments have a better QoL regarding social relations after HA fitting.This is important in this population, since individuals with hearing loss limited to high frequencies mention little difficulty in understanding speech in quiet environments, while in adverse conditions, such as when speech is distorted or in the presence of noise, the person may have many difficulties in speech intelligibility because the number of auditory cues drops considerablyThere are no studies in the literature relating speech perception with QoL. The results of this study allow the conclusion that an open-fit HA improve the speech perception of individuals with hearing loss at high frequencies, both in quiet situations and in competitive noise.The HA fitting improves the QoL of individuals in all areas assessed by the WHOQOL-BREF. Individuals with better speech recognition in noisy environments who use HAs have a better QoL.According to these findings, this research demonstrates the importance of conducting studies to investigate the benefits that amplification can provide for individuals with slope hearing loss, thus improving the decision-making process regarding the prescription of HAs.The acoustic stimulation in hearing loss in high frequencies through the use of OF HA favors the speech recognition and improves the QoL of individuals especially in the social relations domain.


Sensorineural hearing loss is the most common sensory deficit among older adults. Some of the psychosocial consequences of this condition include difficulty in understanding speech, depression, and social isolation. Studies have shown that older adults with hearing loss show some age-related cognitive decline. Hearing aids have been proven as successful interventions to alleviate sensorineural hearing loss. In addition to hearing aid use, the positive effects of auditory training—formal listening activities designed to optimize speech perception—are now being documented among adults with hearing loss who use hearing aids, especially new hearing aid users. Auditory training has also been shown to produce prolonged cognitive performance improvements. However, there is still little evidence to support the benefits of simultaneous hearing aid use and individualized face-to-face auditory training on cognitive performance in adults with hearing loss.This study will investigate whether using hearing aids for the first time will improve the impact of individualized face-to-face auditory training on cognition, depression, and social interaction for adults with sensorineural hearing loss. The rationale for this study is based on the hypothesis that, in adults with sensorineural hearing loss, using hearing aids for the first time in combination with individualized face-to-face auditory training will be more effective for improving cognition, depressive symptoms, and social interaction rather than auditory training on its own.This is a crossover trial targeting 40 men and women between 50 and 90 years of age with either mild or moderate symmetric sensorineural hearing loss. Consented, willing participants will be recruited from either an independent living accommodation or via a community database to undergo a 6-month intensive face-to-face auditory training program (active control). Participants will be assigned in random order to receive hearing aid (intervention) for either the first 3 or last 3 months of the 6-month auditory training program. Each participant will be tested at baseline, 3, and 6 months using a neuropsychological battery of computer-based cognitive assessments, together with a depression symptom instrument and a social interaction measure. The primary outcome will be cognitive performance with regard to spatial working memory. Secondary outcome measures include other cognition performance measures, depressive symptoms, social interaction, and hearing satisfaction.Data analysis is currently under way and the first results are expected to be submitted for publication in June 2018.Results from the study will inform strategies for aural rehabilitation, hearing aid delivery, and future hearing loss intervention trials.ClinicalTrials.gov NCT03112850; https://clinicaltrials.gov/ct2/show/NCT03112850 (Archived by WebCite at http://www.webcitation.org/6xz12fD0B). Hearing loss is a common experience for older adults and is one of the leading causes of nonfatal disease burden for Australians aged 65 years and older ,2. SensoRecent studies have reported that hearing loss among older adults is strongly and independently associated with accelerated cognitive decline -13. EpidIn aural rehabilitation, hearing aid use and auditory training strategies contribute to improving auditory abilities. The basic function of hearing aids is acoustic amplification of sound signals with the aim of restoring the audibility of sounds, thus helping to improve speech perception . StudiesAuditory training is the use of instruction, drill, or practice, designed to increase the amount of information that hearing contributes to a person’s total perception . For exaAlthough the concept of auditory training is not new, its popularity has declined in recent years, and only a small proportion of audiologists (fewer than 10%) offer auditory training to patients with hearing impairment . Also, lStudies have investigated the effects of auditory training with laptops and computers, such as with the Listening and Communication Enhancement (LACE) software, on generalization to speech perception, self-report of communication difficulties, and cognition ,28,33. TResearch has shown that hearing aid devices alone do not always adequately compensate for sensory losses despite significant technological advances in digital technology . TherefoExtending upon preliminary findings ,28,36, tThe study is based on the following hypotheses:In adults with sensorineural hearing loss, hearing aids in combination with face-to-face auditory training will be more efficient for improving cognition than face-to-face auditory training on its own.In adults with sensorineural hearing loss, hearing aids in combination with face-to-face auditory training will be more efficient for improving depression and social interaction than face-to-face auditory training on its own.This study has a randomized crossover trial design. It attained ethics approval on July 22, 2016 (Swinburne’s Human Research Ethics Committee protocol number SHR Project 2016/159).All participants will undergo an individualized face-to-face auditory training program for a period of 6 months and will be randomly allocated to one of the following groups:Group A.Participants who will be fitted with hearing aids only for the first 3 months of the auditory training program—Group B.Participants who will be fitted with hearing aids only for the last 3 months of the auditory training program—Participants will be tested at baseline, and at 3 and 6 months in terms of cognition, depressive symptoms, social interaction, and hearing satisfaction.A crossover design is chosen to allow each participant to serve as their own control . Group AThis study is set in Melbourne, Australia. The study will recruit men and women who are living independently—both in supported independent living accommodation and living independently in the community.To be eligible to participate in the study, participants must satisfy all of the following criteria:Be aged between 50 and 90 yearsHave a good working knowledge of EnglishHave mild (26-40 dB) or moderate (41-70 dB) symmetric sensorineural hearing loss with a pure-tone average threshold of 0.5 to 4 kHz in both earsHave never worn hearing aids previouslyBe willing to wear hearing aids for 3 monthsBe willing to undergo weekly auditory training for a period of 6 monthsBe willing to provide written consent to participate in the studyParticipants will be unable to participate in the study if they have any significant visual impairment that would prevent reading or performing computer-based tasks requiring color recognition. Additionally, study participants with severe or profound hearing loss will not be eligible to take part in the study. Finally, participants with suspected cognitive impairment  will be excluded.Participants will be loaned and fitted with 2 Blamey Saunders hearing aids known as LOF . The hearing aids will be fitted to participants according to the Blamey and Saunders protocol and using the prescription procedures from the National Acoustics Laboratories (NAL) protocol for fitting hearing aids as a guide . ExplanaHistorically, auditory training has been provided in a face-to-face setting that centered on a range of auditory skills, including detection, discrimination, identification, and comprehension. Training often incorporated both drill-like activities, described as analytic therapy activities, and paragraph comprehension activities, which were synthetic in nature. For both activities, the auditory skills that were trained used various stimuli such as syllables, words, phrases, sentences, and continuous discourse .All participants enrolled into the study will undergo weekly individualized face-to-face auditory training for a period of 6 months. Over the 6-month period, each participant will participate in two 12-week individualized speech tracking programs. Participants living in supported independent living accommodation will attend their auditory training sessions at their place of residence, once per week for the 6-month period. Participants living independently in the community will attend their auditory training sessions once per week at Swinburne University of Technology. Each auditory training session will last for approximately 15 min.The type of counseling intervention that will be provided to participants is called Continuous Discourse Speech Tracking . A key aThis program is adopted for this sample population because training materials could be tailored to the personal interests of participants. The materials chosen for the speech tracking program will consist of short stories, which will be long enough to last for a full 12-week program. A new story will be started at the beginning of each 12-week program.The primary outcome measure will be changes in cognitive performance as measured by the spatial working memory component of the Swinburne University Computerized Cognitive Assessment Battery (SUCCAB). Reliability and validity assessment has demonstrated that the SUCCAB, especially the spatial working memory component of this battery, is sensitive to aging and intervention and correlates strongly with memory subsets in the Wechsler Adult Intelligence Scale –Fourth Edition -47.Secondary measures include the other SUCCAB cognition measures, social interaction measured using the Berkman-Syme Social Network Index, and depressive symptoms measured using the Geriatric Depression Scale (GDS). Hearing satisfaction (with or without hearing aids) will be measured using the Abbreviated Profile of Hearing Aid Benefit (APHAB) Inventory.All outcomes will be measured at baseline, after 3 months, and after 6 months .Participant prescreening and assessment will take place at information sessions that will be held at several independent living aged care facilities located in Melbourne and at Swinburne University of Technology. Independent living aged care facilities with existing relationships with Swinburne University of Technology will be chosen. Participants attending information sessions at Swinburne University will be individuals in the community who have expressed interest in assisting with research projects run by the university, and have therefore provided their contact information to be stored in Swinburne’s Centre for Human Psychopharmacology (CHP) database. After providing informed consent, eligible participants will be randomized into 2 equal groups (A and B) for the study, as described in Allowing for 2.5% significance, 80% power, and a moderate effect size (f=0.25), a power analysis indicated that a repeated measures mixed effects design with 3 repeated measures required a total sample size of 34 participants, split evenly between the 2 groups. This allows for the comparison of changes from baseline to 3 and 6 months for the 2 groups. Except for participants from Swinburne’s CHP database who will need to travel to attend their auditory training sessions at Swinburne University, all other participants will attend their appointments at their facilities. As a result, only a 10% allowance was made for attrition resulting in an overall sample size of 40.Aged care facility managers will be contacted by telephone to explain the study. If an aged care facility shows interest in the study, researchers will visit the facility to provide the facility manager with more detailed written and oral information. If the facility manager consents for their facility to participate in the study, the study will be advertised at the facility and promotional materials will be distributed to all the residents, inviting them to an information session. Participants from the CHP database will be contacted by the researchers either by telephone or email to explain the study, and participants who express interest will be invited to attend an information session.At the information session, researchers will explain the purpose and significance of the study. At the same time, a preselection screening will be conducted to identify participants who are willing to wear hearing aids and undergo auditory training to address their hearing loss. Selected participants will be sent a Participant Information and Consent Form package that includes detailed information on the study procedure, a consent form, and a return prepaid envelope. Once written consent is received, participants will be invited to complete baseline measures before enrollment into the study. Recruitment commenced in December 2016.Group A and the other member of each matched pair assigned to Group B. Allocation will be performed using a system of envelopes prepared and opened by the researcher at the time of recruitment.Groups will be matched in terms of the degree of hearing loss (mild or moderate) with 1 member from each matched pair randomly assigned to Given the nature of the intervention, this study will not be blinded as both investigators and participants will know who is wearing hearing aids in each 3-month period.All enrolled participants will not be cognitively impaired and will be screened for adequate cognitive functioning using MMSE. Participants scoring 24 or lower on MMSE will not be eligible for participation.SUCCAB is a validated computer-based cognitive battery consisting of 8 measures that were developed, based on cognitive and neuroimaging literature, to focus on cognitive domains that were most likely to decline with increasing age . StudiesA performance score for each task will be calculated as the ratio of accuracy and reaction time. This approach takes into account variations in accuracy and response time because of speed versus accuracy trade-offs in performance.Participants will undergo the following hearing assessments:Following otoscopy, all participants will undergo tympanometry and acoustic reflex testing to assess the status of the middle ear.To understand the degree of hearing impairment and classify participants according to the type of hearing loss, hearing ability will be measured at threshold frequencies 0.5, 1, 2, 3, 4 kHz in both ears. The choice of frequency to be tested corresponds to the amplification range of most modern hearing aids and is consistent with capturing sensitivity at frequencies affected by sensorineural hearing loss and noise-induced damage. Only participants with either mild or moderate symmetric sensorineural hearing loss will be included in the study.A Web-based Speech Perception Test (SPT) will be used in addition to the standard audiogram for the purpose of measuring hearing loss. SPT is a monosyllabic word test used to characterize the form and degree of hearing loss . There wParticipants will complete a paper-based questionnaire, which will be structured in the following sections:Information on a variety of demographic variables will be collected to describe the characteristics of the study sample.GDS is a self-rating screening scale for measuring levels of depressive symptoms in elderly population . The shoThe Berkman-Syme Social Network Index  will be APHAB  is a selThe following statistical analyses will be performed:Baseline comparison of the 2 groups in terms of demographics, cognition, depression, social interaction, hearing loss, and hearing satisfaction by reporting descriptive statistics of each group as randomized, and an effect size of the difference using Cohen's d.Comparison of the 2 groups in terms of changes in cognition, depression, and social interaction from baseline to 3 months and 6 months, using a per protocol approach for the crossover analysis  and an iAnalysis of SPT results with and without hearing aids as a measure of the efficacy of hearing aids and auditory training with and without hearing aids using multilevel models and again allowing for carryover effects.Analysis of the speech tracking rates from the two 12-week programs of speech tracking using a learning model as described by Blamey and Alcantara . This anAnalysis of the aided and unaided scores from APHAB will be used to assess how the benefit of hearing aids differed between groups and over degree of hearing impairment (mild/moderate hearing loss).This investigation was funded by the Australian Research Council and Blamey and Saunders Hearing Pty Ltd under the Industry Transformation Training Centre Scheme (ARC Project No. IC140100023). The study protocol was reviewed and approved by Swinburne’s Human Research Ethics Committee (SUHREC) on July 22, 2016, protocol number SHR Project 2016/159. The trial is registered in ClinicalTrials.gov with identifier NCT03112850. Recruitment commenced in December 2016 and was completed in June 2017. Researchers obtained written consent from all participants before participating in this trial.The integrity of the trial, including data collection and monitoring, trial progress, adverse events, and compliance with SUHREC reporting procedures will be overseen by the chief (DM) and associate investigators. No serious adverse events are anticipated. The study coordinator (JN) is responsible for communicating protocol changes to relevant stakeholders, including ClinicalTrials.gov registry.Data analysis is currently under way and the first results are expected to be submitted for publication in June 2018.Chronic hearing loss can have a negative impact on several domains of aging such as social engagement, activity, vitality, physical mobility, and cognitive health. Interventions that can significantly delay the onset of sensorineural hearing loss or slow its progression are being actively pursued; however, no disease-modifying treatment is currently available. Understanding the best strategies for aural rehabilitation in older people in whom hearing could compensate for other physical or sensorial limitations may help mitigate cognitive decline.A limitation of the study is that, it will recruit community-dwelling adults who are not cognitively impaired; hence, they may not show improvement in cognitive functioning because of their high baseline scores. However, by focusing on community-dwelling adults, this research will be able to examine the efficacy of programs aimed at minimizing cognitive decline and reducing the rate of transfer to low- and high-care accommodation.For the study intervention, auditory training is being used as the comparator rather than hearing aids, which is popularly known as the main clinical management approach for addressing hearing loss. Although this may be a limitation, the concept of auditory training is not new for addressing hearing loss, as its inception can be traced back to the birth of audiology decades ago, when aural rehabilitation programs were first created for people who had suffered hearing loss . Today,


Cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders. Recently we have generated Cdkl5 KO mice by targeting exon 2 on the C57BL/6N background, and demonstrated postsynaptic overaccumulation of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors in the hippocampus. In the current study, we subjected the Cdkl5 KO mice to a battery of comprehensive behavioral tests, aiming to reveal the effects of loss of CDKL5 in a whole perspective of motor, emotional, social, and cognition/memory functions, and to identify its undetermined roles. The neurological screen, rotarod, hot plate, prepulse inhibition, light/dark transition, open field, elevated plus maze, Porsolt forced swim, tail suspension, one-chamber and three-chamber social interaction, 24-h home cage monitoring, contextual and cued fear conditioning, Barnes maze, and T-maze tests were applied on adult Cdkl5 -/Y and +/Y mice. Cdkl5 -/Y mice showed a mild alteration in the gait. Analyses of emotional behaviors revealed significantly enhanced anxiety-like behaviors of Cdkl5 -/Y mice. Depressive-like behaviors and social interaction of Cdkl5 -/Y mice were uniquely altered. The contextual and cued fear conditioning of Cdkl5 -/Y mice were comparable to control mice; however, Cdkl5 -/Y mice showed a significantly increased freezing time and a significantly decreased distance traveled during the pretone period in the altered context. Both acquisition and long-term retention of spatial reference memory were significantly impaired. The morphometric analysis of hippocampal CA1 pyramidal neurons revealed impaired dendritic arborization and immature spine development in Cdkl5 -/Y mice. These results indicate that CDKL5 plays significant roles in regulating emotional behaviors especially on anxiety- and fear-related responses, and in both acquisition and long-term retention of spatial reference memory, which suggests that focus and special attention should be paid to the specific mechanisms of these deficits in the CDKL5 deficiency disorder.Mutations in the  Cyclin-dependent kinase-like 5 (CDKL5) gene (OMIM #300203) on the chromosome Xp22 region encodes for a serine/threonine kinase, CDKL5 [CDKL5 gene have been identified in patients with severe neurodevelopmental disorders characterized by early-onset intractable epilepsy and severe psychomotor retardation [CDKL5 mutations/deletions are heterozygous females, and are diagnosed as atypical Rett syndrome, West syndrome, or early infantile epileptic encephalopathy 2 (EIEE2) (OMIM #300672) [CDKL5 cause loss-of-function (LOF) of the kinase [The e, CDKL5 . Since 2ardation . The maj#300672) . PathogeN-methyl-D-aspartate (NMDA) receptors in the hippocampus, and regulates seizure susceptibility [Cdkl5 [CDKL5 is expressed widely in most tissues, with highest levels in brain, thymus, and testis . Within tibility . CDKL5 htibility ; amphiphtibility ; Netrin-tibility ; histonetibility ; and Mety [Cdkl5 , 18.in vivo functions and the LOF mechanisms of CDKL5, three independent laboratories including us have generated the Cdkl5 knockout (KO) mice, by targeting exon 6 [Cdkl5 KO mice [In order to elucidate the g exon 6 , 20, exog exon 6 , or exong exon 6 . Moleculg exon 6 ; alteredg exon 6 , 23; abng exon 6 ; selectig exon 6 ; a severg exon 6 ; and a sg exon 6 . We have KO mice .Cdkl5 KO mice. Hyperactivity, impairment in coordination, sociability and memory, and autistic and ADHD-like behaviors have been identified in exon-6-targeted KO mice [Cdkl5 KO mice.Several studies have reported behavioral phenotypes of previously generated  KO mice , 27. Inc KO mice –24. ThusCdkl5 KO mice generated by targeting exon 2 on the C57BL/6N background, which are distinct from the previous mutant mice targeting exon 4 or 6 on the C57BL/6J background. We find that our Cdkl5 KO mice exhibit significantly enhanced anxiety- and fear-related behaviors. Depressive-like behaviors and social interaction of Cdkl5 KO mice are uniquely altered. Both acquisition and long-term retention of spatial reference memory are significantly impaired. These results indicate that CDKL5 plays significant roles in regulating emotional behaviors especially on anxiety- and fear-related responses, and in both acquisition and long-term retention of spatial reference memory, which suggests that focus and special attention should be paid to the specific mechanisms of these deficits in the CDKL5 deficiency disorder.In the current study, we aimed to reveal the behavioral effects of the loss of CDKL5 in a whole perspective of motor, emotional, social, and cognition/memory functions, and to identify its undetermined roles, by adopting a battery of comprehensive behavioral tests to our Cdkl5 -/Y and +/Y mice to a comprehensive battery of behavioral tests   , three-cer test) , and 24-er test) . In the er test) , mean duer test) , number er test) , total der test) , and diser test) . Active traveled  of Cdkl5t result . Then, tt result  and totat result  of Cdkl5Cdkl5 -/Y mice showed a significantly decreased distance traveled , and these enabled Cdkl5 -/Y mice to remember the target hole, as indicated by a progressive reduction in the number of errors Cdkl5 KO CDKL5 mutations are characterized by early-onset intractable epilepsy and severely impaired psychomotor development [Cdkl5 KO mice have been generated by targeting different exons on different backgrounds. To validate an animal model, three criteria are commonly used, i.e. construct validity, face validity, and predictive validity [Cdkl5, the hemizygous male KO mouse models a complete loss of the functional protein. Since most of the human patients are heterozygous female, the KO mouse is not an exact equivalent of the disease model, but it serves as a neurobiological tool to assess the LOF effects of CDKL5 and to find endophenotypes and biomarkers relevant to the human disorder. Regarding the face validity, there are obvious differences in the phenotypes between human patients and KO mice. The most noticeable one relates to the epilepsy. All the reported patients developed severe to moderate epilepsy; however, none of the previously generated KO mice exhibited signs of epilepsy or spontaneous seizures [Cdkl5 KO mice to the human CDKL5 deficiency disorder may be limited, and the predictive value of the current KO mice has to be evaluated by further investigations. Nevertheless, the behavioral studies of Cdkl5 KO mice have provided us with a novel insight into the functions of CDKL5 in specific domains of mammalian behaviors. It is critically important to view the behavioral data in a species-specific context and define the phenotypes in each functional domain.Human patients with elopment , 34. As validity . In the seizures , 19, 21.Cdkl5 KO mice by adopting a battery of comprehensive behavioral tests. Compared with the previous reports of behavioral studies on Cdkl5 KO mice, our study has two distinguishing features. First, we have covered an unbiased, broader range of behavioral repertoires in locomotion, emotion, sociability, and cognition/memory in one study, in order to obtain a behavioral landscape of the animal, and undetermined features of CDKL5 LOF. Second, whereas previous Cdkl5 mutant mice were generated on a C57BL/6J background [In the current study, we revealed behavioral characteristics of ckground –21, our ckground –38. ThesCdkl5 mutant mice, we have confirmed significant deficits in multiple learning and memory tasks in our KO mice. These findings strongly support the fundamental and preserved roles of CDKL5 in cognitive function of mammals. The Cdkl5 KO mice showed impairment in acquisition and long-term retention of spatial reference memory, and in working memory. The spatial reference and working memories are hippocampus-dependent, and long-term retention of spatial reference memory requires interaction between the hippocampus and neocortical regions, such as the entorhinal or perirhinal cortex. Thus the cognitive phenotypes of Cdkl5 KO mice clearly indicate functional impairment in the hippocampus and its associated circuits. In support of this, we and others have identified functional and morphological alteration in the hippocampal neurons of Cdkl5 KO mice, such as disruption in AKT/mTOR-S6/GSK-3β signaling pathways, impairment in dendritic arborization and spine development, and defects in neurogenesis and neuron maturation , thalamus, the bed nucleus of stria terminalis, and the hippocampus [Cdkl5 KO mice have not been conducted yet, above-mentioned data suggest possible involvement of the hippocampus in the etiology of enhanced anxiety and fear. To explain the mechanisms and causal relationship between the enhanced anxiety- and fear-related behaviors of Cdkl5 KO mice, two hypotheses in opposite directions can be constructed as follows.Applying the comprehensive assay strategy, we have identified the significant enhancement of anxiety- and fear- related behaviors in pocampus –50. AlthCdkl5 KO mice have confirmed the postsynaptic overaccumulation of GluN2B in the entire hippocampal formation and the dorsal region by immunoblotting and immunoelectron microscopy respectively, but the ventral hippocampus itself has not been examined. It is necessary to determine the molecular structures and functions of NMDARs in the ventral hippocampus of Cdkl5 KO mice, in order to identify the altered circuits in cognitive and emotional functions.One hypothesis is that the functional impairment in the hippocampus-associated circuits causes an enhanced anxiety, which then leads to an increased fear in a novel environment. The hippocampus is a key structure that regulates both cognition and emotion, and the dorsal and ventral subregions are differently involved in these functions. Cytotoxic lesions of the dorsal hippocampus in rodents impair spatial learning but have no effect on anxiety, whereas lesions of the ventral hippocampus reduce anxiety, without effect on spatial memory . SynaptiThe other hypothesis is that the significantly increased freezing and decreased distance traveled in the altered context represent the overgeneralization of fear memories across context, which then leads to the inappropriate anxiety. Overgeneralization of fear is commonly seen in many mental disorders and conditions, such as specific phobia, obsessive-compulsive disorder, panic disorder, generalized anxiety disorder, and posttraumatic stress disorder (PTSD) , 54. EtiCdkl5 KO mice during conditioning was significantly decreased compared with control mice  and the Animal Research Committee of the National Institute for Physiological Sciences. For the morphological analysis of neurons, mice were euthanized by cervical dislocation. All efforts were made to minimize suffering of mice.Cdkl5 KO mice were generated by targeting the exon 2 as previously described [Cdkl5 KO mouse line with the C57BL/6N genetic background by repeated backcrosses of selected heterozygous females to C57BL/6N males. Some of the backcrosses were performed by a high-speed congenic method using spermatids from immature males (congenic breeding) [ad libitum. All behavioral tests were performed between 9:00 a.m. and 6:00 p.m. After all tests, all apparatus was cleaned with diluted sodium hypochlorite water to prevent a bias due to olfactory cues.escribed . We genereeding) . All behCdkl5 +/Y n = 22, Cdkl5 -/Y n = 22): general health and neurological screening, hot plate test, rotarod test, gait analysis, prepulse inhibition test, light/dark transition test, open field test, elevated plus maze, Porsolt swim test, tail suspension test, one-chamber social interaction test, three-chamber social interaction test, 24-hour social interaction test in home cage, and fear conditioning test; the second group of mice : Barnes maze test and T-maze test. Each behavioral test was separated from each other by at least 1 day. The raw data of behavioral tests, which are not described in this paper, will be disclosed in the gene-brain-phenotyping database (http://www.mouse-phenotype.org/).We prepared two independent groups of mice for behavioral tests. Experiments were done as follows; the first group of mice  as previously reported . Each moA pain sensitivity was measured by the hot plate test according to the previous report . Each MoMotor coordination and motor learning was assessed by the rotarod test . Each moThe gait of each mouse during spontaneous walk/trot locomotion on a translucent plate was shot by a high-speed camera (150 flame/sec.) from underneath. Footprint patterns were analyzed on distance between each stride, variability in stride length, variability around a liner axis, stance width of front paws and hind paws , 66.For the assessment of sensorimotor gating, a startle reflex measurement system  was used to measure startle response and prepulse inhibition as previously described . A mouseThe light/dark transition test was performed as previously described . The appThe open field test was performed as previously described , 68. EacThe elevated plus maze test was performed as previously described , 69. TheThe Porsolt swim test was performed as previously described , 68. TheThe tail suspension test was performed as previously described . Mice weThe social interaction test in a novel environment  was performed as previously described . Two micThe three-chamber social interaction test was performed as previously described . The tesTwenty-four hour monitoring in the home cage was conducted as previously described , 71. TwoFear conditioning tests were performed as previously described . Each moThe Barnes maze test was performed as previously described , 72. TheForced alternation task using the T-maze were performed as previously described with modifications , 74. Thehttps://imagej.nih.gov/ij/), which were modified for each test by Tsuyoshi Miyakawa (available through O’hara & Co.).The applications used for the behavioral studies  were based on the ImageJ program . Data were analyzed by two-tailed t-tests, one-way ANOVA, two-way ANOVA, or two-way repeated-measures ANOVAs. Values in graphs were expressed as mean ± SEM.Cdkl5 -/Y and +/Y mice were euthanized by cervical dislocation. Brains were removed and stained using FD Rapid GolgiStain Kit  as manufacture’s instruction. Two hundred and forty μm thick coronal slices of Golgi-Cox stained brains were prepared. Hippocampal CA1 regions captured by Leica DM6000 B upright microscope using 40x lens were monitored on computer display and traced using the Neurolucida software . Concentric circles (Sholl lines) were drawn at 10 μm increments up to a radius of 180 μm from the center of the soma. Following parameters were measured: (1) the size of cell body, (2) number of dendrites, (3) number of nodes, (4) number of endings, (5) total length of dendrites, (6) each length of dendrites, (7) the number of intersection in every 10 μm from the center of cell body, (8) the number of nodes in every 10 μm from the center of cell body, (9) the number of endings in every 10 μm from the center of cell body. All measurements were performed in a blindfolded manner as to genotypes.Cdkl5 KO mouse line with Thy1-EGFP line M transgenic mouse [Cdkl5 -/Y and +/Y mice were euthanized by cervical dislocation and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains were removed and drop fixed in 4% PFA for 60 min. Fifty μm thick brain slices were cut using the vibrating microslicer DTK-1000 (D.S.K). Apical dendrites of CA1 pyramidal neurons were imaged by the laser scanning confocal microscope FV1000-D  using 60x oil lens (NA 1.35). Image acquisition was performed with 2048 x 2048 pixel size and 0.1 μm slice interval (Voxel size: 0.1 μm3). On the secondary and tertiary branches of CA1 apical dendrites located between 80 and 180 μm from the cell body layers, 6,831 spines from 32 Cdkl5 -/Y neurons and 8,150 spines from 29 Cdkl5 +/Y neurons were analyzed using Imaris 7.4 software . The Imaris FilamentTracer with AutoDepth Method was used to trace dendrites in Surpass view. After centering filaments, Rebuild Dendrite Diameter was performed. Dendrite Threshold  was automatically set. Spine seed point was set to 0.15 μm and maximum length was set to 3 μm. Spine Seed Points Threshold was automatically set and adjusted manually such that all visually discernible spines were detected. Spine Threshold  was set at 3. After rendering, we removed the spines that had 0–30 degrees of Spine Orientation Angle from the Z-axis, because the confocal microscopic images had Z-axis blurring, which might lead to errors in automatic rendering. Finally, Dendrite length, Dendrite Area, Dendrite No. of Spines, Dendrite Spine Density, Spine Length, Spine Max Diameter, Spine Mean Diameter, were automatically obtained from the rendered dendrites and spines. Spine types were classified for 3,442 spines from 15 Cdkl5 +/Y neurons and 4,294 spines from 15 Cdkl5 -/Y neurons, using NeuronStudio software .We crossed the ic mouse . Thy1-GFS1 FigCdkl5 +/Y and Cdkl5 -/Y neurons by Neurolucida. Scale bars = 100 μm.  Representative images of dendrites from Thy1-EGFP positive CA1 pyramidal neurons. Scale bars = 50 μm (B), and 5 μm (C).(A) Representative tracings of dendrites of (EPS)Click here for additional data file.


A subset of tissue samples (8 subjects and 8 controls) were analysed for microRNA expression and the data analysed with mirBase (www.mirbase.org). Using Multivariate statistical analysis, Ingenuity pathway analysis software, DIANALAB database and published literature, the association of significant metabolites with relevant cellular pathways were understood. Higher concentrations of glutamate, taurine, myo-inositol, creatine and inosine were present in aqueous extracts and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in lipid extracts in the VV group compared with non-VV group. Out of 7 differentially expressed miRNAs, spearman correlation testing highlighted correlation of hsa-miR-642a-3p, hsa-miR-4459 and hsa-miR-135a-3p expression with inosine in the vein tissue, while miR-216a-5p, conversely, was correlated with phosphatidylcholine and phosphatidylethanolamine. Pathway analysis revealed an association of phosphatidylcholine and sphingomyelin with inflammation and myo-inositol with cellular proliferation.Metabolic phenotypes reflect both the genetic and environmental factors which contribute to the development of varicose veins (VV). This study utilises analytical techniques to provide a comprehensive metabolic picture of VV disease, with the aim of identifying putative cellular pathways of disease pathogenesis. VV (n = 80) and non-VV (n = 35) aqueous and lipid metabolite extracts were analysed using 600 MHz  However, how disease variants of risk loci disrupt the biochemistry of the cell and how these events translate into the formation of the varicose vein is still not known. Understanding how biochemical aberrations modulate metabolic pathways, enzymatic activity and normal cellular function is the natural next step in the investigation of the disease.Primary varicose vein disease, a vascular condition characterised by elongated, dilated and tortuous veins in the lower limbs, remains one of the most commonly reported chronic conditions in western medicine. While recent studies have identified familial clustering of disease, and a genetic component is generally accepted, age, occupation, pregnancy and obesity are still key risk factors. The determination of the existence of mechanistically different varicose vein “sub-phenotypes” would be important as this could reflect the different effects that various environmental factors have on disease progression. The morphological characteristics of the disease have been well documented, and known histological changes in the dilated vein include intimal hyperplasia and disruption of smooth muscle cells and the extracellular matrix3. The metabolic profiles of human varicose veins have been previously shown to be different from healthy vein tissue, when analysed using magic angle spinning (MAS) 1H- nuclear magnetic resonance (NMR) spectroscopy. 1H-MAS NMR spectroscopy allows for the profiling of intact whole tissue without the need for chemical extraction, and also provides information on metabolite mobility and metabolite localisation within the tissue4. Lactate, myo-inositol and creatine were found to be the differential biomarkers that distinguished between the healthy and diseased vein groups, and triglyceride moieties were also observed to be lower in concentration in varicose tissue5. However, the study in question only utilised one profiling technique and a very small sample size. Therefore, to be able to attain a more comprehensive metabolic picture, and move beyond phenotypic observation to identification of potential mechanisms, analysis of extracted metabolites from a larger number of participants is required. In addition to using multiple analytical platforms such as NMR and ultra performance liquid chromatography coupled to -mass spectrometry (UPLC-MS), microRNAs (miRNAs) have been profiled for a subset of patients to explore modulated pathways. miRNAs are small 22 nucleotide RNAs, that play an important role in cell regulation by silencing genes through targeting mRNAs for cleavage. miRNAs are involved in the regulation of many key cellular pathways and as such have been identified as potential markers for patient stratification and as therapeutic targets. Additionally, UPLC-MS, an inherently more sensitive analytical technology, can complement NMR analysis and provide further information of the perturbation of low concentration metabolites, and extend profiling coverage. This study uses three analytical techniques including NMR, UPLC-MS and miRNAs to provide a comprehensive metabolic picture of varicose vein disease, aiming to synthesise a metabolic map of the disease and identify putative cellular pathways of disease pathogenesis. Aqueous extracts from humans’ varicose vein samples and humans’ non-varicose vein control were analysed using NMR and UPLC-MS HILIC profiling. Lipid extracts from the diseased and controls veins samples were analysed using UPLC-MS. miRNAs profiling was performed on randomly selected diseases and long saphenous vein controls samples.Metabonomics employs established and reproducible tools for the identification of the metabolic status of an individual and are highly appropriate for the evaluation of the biochemical components of a disease phenotypeInitial principal component (PC) modelling of the NMR data showed some stratification in the data . The significant peaks highlighted as driving the group differences corresponded to signatures from myo-inositol, taurine and glutamic acid (glutamate) which were higher in the disease group  was carried out using a selected discriminatory peak as a driver for calculation of the correlation matrix6. Figure myo-inositol (δ4.07), although at δ3.269, the taurine peaks and myo-inositol peaks are overlapping.The OPLS-DA models produced with the aqueous extract 2Y = 0.486 and R2Y = 0.693  experimental run was indicated by the tight clustering of the QC samples near the middle of statistical space in the PCA scores plot from the multivariate analysis of both the electrospray ionization (ESI)+ and ESI- UPLC-MS data . The metabolites identified as driving the class differences, again using the loadings S-plot, included phosphatidylserines, phosphatidylcholines, sphingomyelins, ceramides, phosphatidylethanolamines and phosphatidylinositols -trisphosphate (PIP3) to phosphatidylinositol 4,5-bisphosphate (PIP2), are predicted to contribute to the regulation of both the PI3K/AKt and MAPK pathways, which are known to be involved in cell proliferation. The identities of remaining nodes of the pathway are defined in the supplementary section.A Spearman correlation matrix was constructed to investigate the correlation between miRNA expression and the levels of the significant metabolites identified through the analytical chemistry. The resultant heatmap and dendrogram are provided Fig. . This himyo-inositol, taurine and inosine were present in higher concentrations in varicose veins. The validity of these results was reenforced by the agreement of both MS-based analytical techniques and the results of the NMR spectroscopy in identification of the differential metabolites in varicose vein disease. In addition the non-polar metabolites sphingomyelin and several phospholipids were also found to be elevated in the disease group. Pathway analysis provided a potential network of molecular interactions between these metabolites and identified miRNAs within VV; this network paves the way for further investigation of disease etiology. Variations in age, sex, differences in past medical history and medications had no correlation with any metabolic differences noticed between the groups.The aim of this study was to provide a comprehensive characterisation of the metabolic profiles of varicose and non-varicose veins, and to identify any metabolic differences between the two. Chemometric analysis of the tissue aqueous extracts revealed that the polar metabolites creatine, glutamate, 7. In adition glutamate is also involved in the production of gamma-aminobutyric acid (GABA) in the GABA-shunt and also part of glutathione synthesis8. Glutathione, being one of the primary conjugative molecules of phase two detoxification, is an important factor in the cellular defence mechanism and also helps regulate cell proliferation and apoptosis9. However there were no discernable changes in glutathione levels detected in the samples.Glutamate, one of the key metabolites identified by the multivariate modeling, is an alpha-amino acid, a key metabolite in many aspects of cellular metabolism, and plays a central role in hepatic amino acid metabolism, glycolysis, gluconeogenesis and the tricarboxylic acid cycle10. Administration of taurine has also been shown to protect from stress-induced vascular endothelial cell apoptosis11, and high levels of taurine are thought to promote cell survival.Another of the significantly changed metabolites is taurine. Taurine, a ubiquitous organic osmolyte, is one of the most abundant amino acids within the body, although it is not involved in protein synthesis. Metabolically derived from cysteine, taurine is involved in the formation of bile salts, regulation of cell volume and modulation of intracellular calcium concentration, essential for the function of signal transduction pathwaysMyo-inositol, one of the more abundant inositol isomers, is a carbocyclic polyol and is also a structural component of a number of secondary cellular messengers such as inositol phosphates, phosphatidylinositol and phosphatidylinositol phosphate lipids. Myo-inositol, like taurine, predominately operates as an intercellular osmolyte, and is also involved in the Akt signalling pathway, membrane trafficking and regulating apoptosis. Cytosolic myo-inositol is essential for cell survival and promotes growth and proliferation12.et al. using matrix assisted laser desorption ionization imaging13. Sphingomyelin is the most abundant type of sphingolipid and constitutes a key structural component of cellular membranes and vesicles. Sphingomyelin acts as a platform for the regulation of transmembrane signalling, and also intracellular vesicle trafficking as bilayer sphingomyelin levels are involved in chemotaxis-based migration. This modulation extends to transferrin-mediated cell proliferation triggering cell growth and division14.Sphingomyelin was elevated in VV tissue, and correlation analysis showed its concentration is closesly linked to the hsa-miR-142-5p part of the predicted VV network  is related to hypoxia, as the gene encoding for CKa includes a hypoxia induced element in the promoter region16; this means that during hypoxic conditions, cells will increase choline uptake potentially triggering downstream cell cycle progression. Phosphatidylcholine is also a substrate for phospholipase A2 (PLA2). PLA2 causes hydrolysis of phosphatidylcholine into free fatty acids such as arachidonic acid (AA) and lysophosphatidylcholine. AA is a precursor molecule for prostaglandins and leukotriene, both responsible for chronic inflammation13. We didn’t detect phosphatidylcholine with AA incorporated, however, we detected phosphatidylcholine (20:3).The statistical analysis showed that there is an increase of cellular concentrations of a wide range of phospholipids which are the key component of the lipid bilayer and are essential for the viability of eukaryotic cells. While both phosphatidylserine and phosphatidylethanolamine, have been shown to be involved in intracellular signal transduction and cell proliferation, of most interest is phosphatidylcholine. Phosphatidylcholine, a glycerophospholipid, is synthesised by the CDP-choline pathway or through the hepatic phosphatidylethanolamine methyltransferase. As a key component of membranes and lipid messengers, phosphatidylcholine is directly involved in cell cycle progression and cell proliferation17 and increased creatine has been seen in dilated cardiomyopathy18. Correlation matrix analysis , the activity of which is closely linked with local pH and oxygen levels21; Indeed studies investigating the effect of sphingomyelinase deficiency has shown that cells that do not catabolise sphingomyelin efficiently are protected from stress-induced apoptosis22. Each player in this complex set of interactions has been shown to be a bioactive lipid, with sphingomyelin, phosphocholine, phosphatidylcholine, ceramide and DAG all exerting an influence on the survivability of the cell, albeit an opposing one. The first three seemingly are encouraging growth and survival, whereas the latter two promoting cell death and quiescence23. Taken together this could indicate that the local chemical environment and physical duress which cells are exposed to following chronic blood stasis could be triggering changes in the balance of metabolism and disrupting lipid homeostasis, leading to the production of certain pro-survival molecules with the relevant downstream effects.A similar narrative can be discerned from evaluating the results while focusing on sphingolipid metabolism. Sphingolipids share a common 18 carbon backbone, with different accessory structures giving rise to the various members of the sphingolipid family. The metabolic network could be thought of a web of interconnected pathways, branching out from a common progenitor molecule and coming together to terminate in a common catabolic end-point. The various intermediate molecules on each branch of the network, however, have highly diverse functions, and as such the metabolic trajectory each molecule is highly regulated to ensure correct cellular function. Sphyngomyelin, mentioned above and found to be raised in varicose veins, is synthesised by the transfer of a phosphocholine group from phosphatidylcholine  to ceramide, by sphingomyelin synthases (SMSs)This study has presented a metabolic characterisation of the VV disease, highlighting the molecular processes underlying VV pathogenesis and progression. It has been well established that such a phenotypic switch will affect vein wall contraction and result in vein wall relaxation and weakness, which may be the cause of the tortuous and loose appearance of the diseased vein. The identified metabolites suggest that the processes of cell proliferation and survival should be further investigated with a view to identifying potential therapeutic drug targets. Development of drug therapy in venous disease may help in halting the disease progression as well as a preventative measure for individuals with a genetic propensity for the development of the disease.24 and neither parent drugs nor their metabolites were detected in our NMR or LCMS based profiling, it is possible that the above factors confounded the presented results by pharmacological effect. It is, nonetheless, important to stress that despite these issues the varicose vein samples were consistently different from the control groups in the statistical modelling, suggesting that any variability within the non-varicose veins groups was far outweighed by the differences between the two groups. Ultimately due to the ethical issues surrounding vein sample retrieval from often young and otherwise healthy individuals, it may prove challenging if not impossible to correctly age match groups in future varicose veins studies.One of the key limitations of the work presented here is the heterogeneity of the control group. The control group did not accurately match with the varicose veins group in terms of demographic features, past medical history and the medications. Factors such as age, medications and past medical history may influence the metabolic profile of vein tissue. However, to our knowledge, there has been no demonstration of the effect of age on metabolic profile of vein tissue so far. The control group included great saphenous vein samples from patients undergoing lower limb bypass operations or amputations in addition to vein samples from other anatomical locations such as the neck and lower abdomen, which are subject to different biomechanical stresses than the GSV. While statins and most other cardiovascular medications have a short half-life (less than 24 hours)25, and how CEAP grading relates to metabolic phenotypes. Future studies should potentially explore any differences of metabolites in tissue, serum and urine between patients of different disease grades. This could aid clinicians in following disease progression and help tailor therapy accordingly. Varicose vein disease is a multifactorial disease influenced by both environmental factors such as occupation, obesity, pregnancy and also has a strong familial predisposition. Environmental stimuli in the patient exposes the vein wall to high pressure and hypoxia, which in combination trigger molecular changes in the vein wall leading to vein wall relaxation and dilatation. This work has shown that the metabolic profiles of human varicose veins are systematically different from non-varicose veins tissues, as well as exemplifying the utility of the metabonomic approach in understanding the molecular changes at cellular level of vascular disease.Another consideration for the future is CEAP classification, the scheme by which the disease is classified into different grades based on severity and type of presentationEthical approval was obtained for the research from the Riverside Research Ethics Committee (RREC 3092). The research has also been registered to the Imperial College London Research Services, and the Imperial College Healthcare NHS Trust Research and Development department and all the experiments were performed in accordance with the department’s guidelines and regulations. All the specimens collected were anonymised according to the rules set out in the Human Tissue Act (2004). Informed consent was obtained from all the patients involved in the study. Varicose veins were retrieved from patients who were treated with ligation of sapheno-femoral junction and stripping of great saphenous vein (GSV) or phlebectomies within Imperial College London Healthcare NHS Trust. Initial assessment of the patient was performed in outpatient department. Duplex ultrasound imaging of the lower limb venous system was performed for all varicose veins patients by accredited vascular scientists where the presence of venous reflux was assessed. Control veins were obtained from patients undergoing any surgery which involved the routine removal of non-varicose veins that would otherwise be discarded, including residual vein following vein harvest for extremity arterial bypass. Patients with a current or past history of varicose veins were not included in the non-varicose vein control group. Varicose and non-varicose veins of the affected limbs of patients with a history of deep vein thrombosis (DVT) or superficial thrombophlebitis were excluded from the study. Patients with congenital diseases associated with varicose veins, including Klippel-Trenaunay Syndrome, arterio-venous malformations, secondary varicose veins, or human immunodeficiency virus (HIV) infection were not included in the study.In total, 80 participants with varicose veins and 35 with non-varicose vein samples were included in the study. Tissue and demographic details are provided in Supplementary Table The organic solvents used were chloroform, acetonitrile, DCM, ISP, hexane and MTBE . Methanol (Fisher) and water were (Fluka) both LC-MS grade. Additionally, formic acid, leucine enkephalin, ammonium formate (HPLC grade) and sodium formate, were obtained from Sigma-Aldrich, Gillingham, UK. Deuteriated chloroform with 0.05% tetramethylsilane (TMS) was obtained from Goss Scientific, UK. Biological tissue was handled in Class II biological cabinet and local laboratory protocols were strictly followed. Chloroform was handled in safety cabinet due to hazardous risks involved with chloroform. Local laboratory safety precautions were followed while handling liquid nitrogen.26. Aqueous and organic extractions were performed sequentially on the tissue samples. Organic extraction was performed first followed by aqueous extraction26 (please see Supplementary Information for the details of extraction protocol).Metabolite extraction was performed as previously optimised in human vein tissueet al.26 (please see Supplementary information for the details of aqueous extracts preparation). Aqueous vein extracts were analysed using 1H NMR spectroscopy at a field strength of 14.1 T (1H 600.29 MHz) using a 5 mm broadband inverse configuration probe with a z axis magnetic field-gradient capability. The spectrometer was equipped with a Bruker Sample Jet system set to 5 mm shuttle mode with a cooling rack of refrigerated tubes at 6 °C and controlled via a Bruker Avance III console . A standard 1D pulse sequence  propionate (TSP) at δ1H 0.00 in TOPSPIN 3.0 software . The baseline was corrected manually. Spectra were imported into MATLAB R2009b  using an in-house algorithm built in MATLAB. The spectral region containing the water resonance (from δ1H 4.68 to 5.24) was removed from all spectra. In addition, the resonance of TSP (from δ1H −1 to 0.2) was removed from all spectra. All spectra were subsequently aligned and normalised using probabilistic quotient normalisation27. NMR assignments were performed by observing signal peak, chemical shift and comparing this to a list of standards available on central database HMDB, and an internal database. Statistical Total Correlation Spectroscopy (STOCSY)6, which computes the correlation between variables in the spectrum, by taking advantage of the multicollinear nature of NMR data, was also employed for the identification of related peaks, aiding the metabolite identification process.Two aliquots holding dried aqueous extracts from 80 varicose vein and 35 non-varicose vein samples were prepared for NMR spectroscopy using the method previously by Anwar 28. A total of 50 µl from each sample was pooled to make a quality control (QC) sample29. QC samples were injected every 10 samples throughout the run to monitor the stability of the UPLC-MS instrument. Details of samples preparation for UPLC-MS analysis is given in supporting information section. Organic extracts (lipid profiling) from 80 varicose vein and 35 non-varicose vein samples were analyzed as previously described28 using an Acquity UPLC system  coupled to a Q-TOF Premier mass spectrometer . For chromatography of organic extracts, an Acquity UPLC column CSH  was used. Analysis of aqueous extracts from 80 varicose vein and 24 non-varicose vein samples with hydrophilic interaction liquid chromatography (HILIC) was performed as previously described28 using an Acquity UPLC System , coupled with LCT Premier mass spectrometer . An Acquity UPLC BEH HILIC column  was used and maintained at 35 °C. Details of instruments settings 28.UPLC-MS analysis was performed with the protocols previously published by our group30 (version 1.46.0) in the R programing language (version 3.2.0). After peak-picking, retention time correction and grouping of the chromatographic peaks, an algorithm was used for filling zero values using the adjacent background intensity. The three-dimensional table produced consisted of features characterized by their m/z, retention time and signal intensity. The dataset was then subjected to total area normalization.For UPLC-MS, data were processed using the XCMS packagehttp://www.hmdb.ca) and LIPID MAPS, (http://www.lipidmaps.org), 2) isotopic patterns, 3) in-house developed libraries of standards28 and 4) MSE and/or MS/MS spectra, by matching to tandem MS experiments from online databases.Metabolite structural assignments for UPLC-MS were conducted by: 1) matching accurate mass measurements to theoretical values from on-line databases including METLIN (http://metlin.scripps.edu/metabolites), HMDB  was used to analyse the data acquired from UPLC-MS, whereas the NMR data was analysed using MATLAB R2009b  with scripts developed in-house. Both principal components analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA) were performed. The validity of the OPLS models were assessed by performing 1000 permutations with randomized scrambling of class labels. OPLS loadings S-plots derived from the OPLS-DA models were used to discern the variables driving group differences. Further univariate analysis of variance (one-way ANOVA) was performed on the complete set of data to identify the individual effects of each variable; Benjamini-Yekutieli correction was used to adjust the significance statistics to account for multiple testing2Y, R2X values and p values  for each model were calculated.A multivariate partial least square (PLS) regression modelling approach was used to identify any influences of demographic and clinical confounders Table  on the bA subset of tissue samples (8 test subjects and 8 controls) were analysed for microRNA expression, by Miltenyi Biotec . These were chosen randomly from the cohort of 115 samples prepared for metabonomic analysis. Samples underwent RNA extraction using TRIzol (Invitrogen) under RNase-free conditions. Details of experimental protocol are given in supplementary information section. The quantity and quality of the RNA extraction was investigated by bioanalyzer and the generation of an RNA Integrity Number (RIN). A RIN value of greater than 6 was considered to be acceptable and based on this six samples (3 test and 3 control) of the original 16 samples were rejected. Electropherograms of the accepted samples indicated that there was good signal intensity in each of the sample runs. The fold change ratios of expression were calculated based on light intensity (light units) after hybridisation of the extracted RNA onto microarrays.www.mirbase.org), after which the miRNAs were ranked according to experimental versus control expression ratios. The 10 most correlated and the 10 most anti-correlated miRNAs were then examined for their expected regulated cellular processes using the DIANALAB database, which provided a list of potential KEGG pathways, ranked by statistical confidence. Prediction modelling tool TargetScan was then used to produce a list of mRNAs under the regulation each of the target miRNAs, which was subsequently imported into the PANTHER classification system to produce a list of pathways containing the mRNAs in question. These pathways were analysed using a statistical overrepresentation analysis to produce a p-value to identify which ones were statistical significant for the experiment. All processes that were statistically significant were incorporated into Ingenuity Pathway Analysis  to create a network biology system for the interaction between both the miRNAs and the metabolites identified from the metabonomic analysis. A Pearson correlation matrix was constructed to identify which miRNA expression levels correlated with concentrations of the previously identified significant metabolites. Finally, the results from all the above analyses were combined into a single IPA pathway mapping analysis, which mapped the biological functions of the differential metabolites and miRNAs.Functional analysis of the identified miRNA targets was performed using computational methods. Initial confirmation of miRNA nomenclature was achieved using miRNA database mirBase (SI