CRISPR/Cas9 mediated generation of an ovine model for infantile neuronal ceroid lipofuscinosis (CLN1 disease).

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating monogenetic lysosomal disorders that affect children and young adults with no cure or effective treatment currently available. One of the more severe infantile forms of the disease (INCL or CLN1 disease) is due to mutations in the palmitoyl-protein thioesterase 1 (PPT1) gene and severely reduces the child's lifespan to approximately 9 years of age. In order to better translate the human condition than is possible in mice, we sought to produce a large animal model employing CRISPR/Cas9 gene editing technology. Three PPT1 homozygote sheep were generated by insertion of a disease-causing PPT1 (R151X) human mutation into the orthologous sheep locus. This resulted in a morphological, anatomical and biochemical disease phenotype that closely resembles the human condition. The homozygous sheep were found to have significantly reduced PPT1 enzyme activity and accumulate autofluorescent storage material, as is observed in CLN1 patients. Clinical signs included pronounced behavioral deficits as well as motor deficits and complete loss of vision, with a reduced lifespan of 17 ± 1 months at a humanely defined terminal endpoint. Magnetic resonance imaging (MRI) confirmed a significant decrease in motor cortical volume as well as increased ventricular volume corresponding with observed brain atrophy and a profound reduction in brain mass of 30% at necropsy, similar to alterations observed in human patients. In summary, we have generated the first CRISPR/Cas9 gene edited NCL model. This novel sheep model of CLN1 disease develops biochemical, gross morphological and in vivo brain alterations confirming the efficacy of the targeted modification and potential relevance to the human condition.

Interactive binocular treatment (I-BiT) for amblyopia: results of a pilot study of 3D shutter glasses system.

PURPOSE: A computer-based interactive binocular treatment system (I-BiT) for amblyopia has been developed, which utilises commercially available 3D 'shutter glasses'. The purpose of this pilot study was to report the effect of treatment on visual acuity (VA) in children with amblyopia. METHODS: Thirty minutes of I-BiT treatment was given once weekly for 6 weeks. Treatment sessions consisted of playing a computer game and watching a DVD through the I-BiT system. VA was assessed at baseline, mid-treatment, at the end of treatment, and at 4 weeks post treatment. Standard summary statistics and an exploratory one-way analysis of variance (ANOVA) were performed. RESULTS: Ten patients were enrolled with strabismic, anisometropic, or mixed amblyopia. The mean age was 5.4 years. Nine patients (90%) completed the full course of I-BiT treatment with a mean improvement of 0.18 (SD=0.143). Six out of nine patients (67%) who completed the treatment showed a clinically significant improvement of 0.125 LogMAR units or more at follow-up. The exploratory one-way ANOVA showed an overall effect over time (F=7.95, P=0.01). No adverse effects were reported. CONCLUSION: This small, uncontrolled study has shown VA gains with 3 hours of I-BiT treatment. Although it is recognised that this pilot study had significant limitations-it was unblinded, uncontrolled, and too small to permit formal statistical analysis-these results suggest that further investigation of I-BiT treatment is worthwhile.

Investigation of the relationship between transcranial impedance and intracranial pressure.

Studies on piglets have shown that cranial bioimpedance (Z) measurements correlate well with invasively measured intracranial pressure (ICP). We have tested the feasibility of collecting transcranial impedance from a clinical device for measuring whole-body water content (ImpediMed SFB7). In the clinical study, 50 normal healthy volunteers had transcranial impedance measured using nine different head montages (forehead to mastoid (left/right), temporal to mastoid (left/right), forehead to temporal (left/right), forehead to occipital (left/right) and temporal to temporal). Impedance was measured 20 times over a frequency range per montage and ANOVA used to test for effects of electrode position upon recorded value. For the experimental study, five sedated and ventilated Marino sheep were instrumented for intraventricular ICP and transcranial impedance measurement. Measures of ICP were recorded while ICP was increased from baseline to greater than 50 mmHg in five steps using an intraventricular infusion of mock CSF. There is a significant effect of electrode position and gender upon transcranial impedance (p < 0.001). The temporal-mastoid electrode position had significantly lower impedance values in keeping with its shorter path length. ICP correlated with craniospinal compliance measurements and Impedance vs Freq by ICP step shows a clear ICP dependence (p = 0.007) across the sheep.

Reducing the oxygen concentration of gases delivered from anaesthetic machines unadapted for medical air.

High fractional concentrations of inspired oxygen (FiO(2)) delivered over prolonged periods produce characteristic histological changes in the lungs and airway of exposed animals. Modern medical anaesthetic machines are adapted to deliver medical air (FiO(2)=0.21) for the purpose of reducing FiO(2); anaesthetic machines designed for the veterinary market have not been so adapted. Two inexpensive modifications that allow medical air to be added to the gas flow from veterinary anaesthetic machines are described. The advantages and disadvantages of each modification are discussed.

Effect of chest wall vibration timing on peak expiratory flow and inspiratory pressure in a mechanically ventilated lung model.

OBJECTIVE: To investigate the effects of chest wall vibration timing on air flow and pressure in a ventilated lung model. DESIGN: Laboratory-based bench study. PARTICIPANTS: Thirty physiotherapists with experience in intensive care. INTERVENTION: Physiotherapists applied three sets of eight chest wall vibrations to an intubated, mechanically ventilated mannequin. Vibrations were applied at the start of expiration (optimal), mid to late inspiration (early) and early to mid expiration (late). Air flow, peak pressure and volume were measured continuously. Forces applied during vibrations were recorded using a force-sensing mat, placed under the physiotherapists' hands. RESULTS: During optimal and early vibrations, peak expiratory flow increased significantly compared with baseline ventilation [mean difference for optimal vibrations 8.8l/minute, 95% confidence interval (CI) 6.0 to 11.6; mean difference for early vibrations 7.0l/minute, 95% CI 4.3 to 9.9]. Late vibrations did not enhance expiratory flow. Peak inspiratory pressure was significantly higher during early vibrations compared with baseline values (mean difference 5.6cmH(2)O, 95% CI 2.9 to 8.2). Peak inspiratory pressure generated during early vibrations was, on average, 8.4cmH(2)O greater than with optimal timing. CONCLUSION: The safety and effectiveness of respiratory physiotherapy treatments are likely to be influenced by the timing of vibrations within the breath cycle. Early vibrations generate potentially dangerous peak inspiratory pressures. Late vibrations, although not harmful, are not effective at increasing peak expiratory flow. This is an important consideration when training physiotherapists and evaluating outcomes of treatments in intensive care.

Second-eye cataract surgery in elderly women: a cost-utility analysis conducted alongside a randomized controlled trial.

AIM: The aim of this study was to evaluate the cost-effectiveness of second-eye cataract surgery for older women with minimal visual dysfunction in the eye to be operated on from a Health and Personal Social Services perspective, compared to waiting list controls who had already undergone first-eye cataract surgery. METHODS: A cost-utility analysis was undertaken alongside a randomized controlled trial of second-eye cataract surgery in secondary care ophthalmology clinics. A total of 239 women over 70 years old with one unoperated cataract were randomized to cataract surgery (expedited, approximately 4 weeks) or control (routine surgery, 12 months wait). Outcomes were measured in terms of quality-adjusted life years (QALYs), with health-related quality of life estimated using the EuroQol EQ-5D. RESULTS: The operated group had costs which were, on average, pound646 more than the control group (95% confidence interval, pound16-1276, P<0.04) and had a mean QALY gain of 0.015 (95% confidence interval, -0.039 to 0.068, P=0.59) per patient over 1 year. Therefore, the incremental cost-utility ratio was pound44,263 over the 1-year trial period. In an analysis modelling costs and benefits over patients' expected lifetime, the incremental cost per QALY was pound17,299, under conservative assumptions. CONCLUSIONS: Second-eye cataract surgery is not likely to be cost-effective in the short term for those with mild visual dysfunction pre-operation. In the long term, second-eye cataract surgery appears to be cost-effective unless carer costs are included.

Radiological classification of optic pathway gliomas: experience of a modified functional classification system.

Optic pathway gliomas (OPGs) in childhood are associated with neurofibromatosis type 1 (NF1) and since 1958 have been classified anatomically using the Dodge classification (DC). MR scanning permits a more detailed anatomical description than can be classified by this historical system. A modified Dodge classification (MDC) has been applied to MRI scans from a cohort of 72 patients (36.1% NF1-positive) from 4 centres participating in an international clinical trial. The MDC was feasible, applicable and more detailed than the original DC. NF1-positive cases more commonly involved both optic nerves (p = 0.021) and other multiple locations (p = 0.001). NF1-negative tumours more commonly involved the central chiasm (p = 0.005) and hypothalamus (p = 0.003). Fewer hypothalamus-positive tumours were associated with optic nerve involvement (p = 0.009), whereas more were associated with central chiasm involvement (p<0.001). From diagnosis to follow-up, there was concordance between DC and MDC in 51/72 cases (70.8%). The MDC is therefore proposed for use in clinical trials of new treatments for OPGs.

Simultaneous measurement of force and respiratory profiles during chest physiotherapy in ventilated children.

There are currently no objective means of quantifying chest wall vibrations during manual physiotherapy. The aims of the study were to (i) develop a method to quantify physiotherapy-applied forces and simultaneous changes in respiratory flow and pressure, (ii) assess the feasibility of using this method in ventilated children and (iii) characterize treatment profiles delivered by physiotherapists in the paediatric intensive care unit. Customized sensing mats were designed and used in combination with a respiratory profile monitor. Software was developed to align force and flow data streams. Force and respiratory data were successfully collected in 55 children (median age 1.6 years (range 0.02-13.7 years)). Physiotherapists demonstrated distinctive variations in the pattern of force applied and manual lung inflations. The maximum applied force ranged from 15 to 172 N, and was correlated with the child's age (r = 0.76). Peak expiratory flow increased significantly during manual inflations both with and without chest wall vibrations (p < 0.05). This method provides the basis for objective assessments of the direct and independent effects of vibration forces and manual lung inflations as an essential precursor to developing evidence-based practice.

Preliminary results from the use of the novel Interactive binocular treatment (I-BiT) system, in the treatment of strabismic and anisometropic amblyopia.

BACKGROUND: We have developed a novel application of adapted virtual reality (VR) technology, for the binocular treatment of amblyopia. We describe the use of the system in six children. METHODS: Subjects consisted of three conventional treatment 'failures' and three conventional treatment 'refusers', with a mean age of 6.25 years (5.42-7.75 years). Treatment consisted of watching video clips and playing interactive games with specifically designed software to allow streamed binocular image presentation. RESULTS: Initial vision in the amblyopic eye ranged from 6/12 to 6/120 and post-treatment 6/7.5 to 6/24-1. Total treatment time was a mean of 4.4 h. Five out of six children have shown an improvement in their vision (average increase of 10 letters), including those who had previously failed to comply with conventional occlusion. CONCLUSIONS: Improvements in vision were demonstrable within a short period of time, in some children after 1 h of treatment. This system is an exciting and promising application of VR technology as a new treatment for amblyopia.

Modified virtual reality technology for treatment of amblyopia.

PURPOSE: The conventional patching/occlusion treatment for amblyopia sometimes gives disappointing results for a number of reasons: it is unpopular, prolonged, frequently resulting in poor or noncompliance, and also disrupts fusion. The aim of this research was to develop a novel virtual-reality (VR)-based display system that facilitates the treatment of amblyopia with both eyes stimulated simultaneously. METHODS: We have adopted a multidisciplinary approach, combining VR expertise with a team of ophthalmologists and orthoptists to develop the Interactive Binocular Treatment (I-BiT) system. This system incorporates adapted VR technology and specially written software providing interactive 2D and 3D games and videos to the patient via a stereo (binocular) display, and a control screen for the clinician. RESULTS: We developed a prototype research system designed for treatment of amblyopia in children. CONCLUSIONS: The result is a novel way to treat amblyopia, which allows binocular treatment. It is interactive, and as it is partially software based, can be adapted to suit the age/ability, and needs of the patient. This means that the treatment can be made captivating and enjoyable. Further research is on-going to determine the efficacy of this new modality in the treatment of amblyopia.

An oral carcinogenicity and toxicity study of senna (Tinnevelly senna fruits) in the rat.

Senna (Tinnevelly senna fruits), a known laxative derived from plants, was administered by gavage to Sprague-Dawley (Crl:CD (SD) BR) rats once daily at dose levels of 0, 25, 100 and 300 mg/kg/day for up to 104 consecutive weeks. Based upon clinical signs related to the laxation effect of senna, the highest dose (300 mg/kg/day) was considered to be a maximum tolerated dose. Sixty animals per sex were assigned to the control and dose groups. Assessments included clinical chemistry, hematology, full histology (control and high-dose groups; in addition, low and mid dose: intestinal tract, adrenals, liver, kidneys, brain and gross lesions) and toxicokinetics. The primary treatment-related clinical observation was mucoid feces seen at 300 mg/kg/day. When compared to controls, animals administered 300 mg/kg/day had slightly reduced body weights, increased water consumption and notable changes in electrolytes in serum (increases in potassium and chloride) and urine (decreases in sodium, potassium and chloride). The changes in electrolytes are most likely physiologic adaptations to the laxative effect of senna. At necropsy, dark discoloration of the kidneys was observed in animals in all treated groups. Histological changes were seen in the kidneys of animals from all treated groups and included slight to moderate tubular basophilia and tubular pigment deposits. In addition, for all treated groups, minimal to slight hyperplasia was evident in the colon and cecum. These histological changes, together with the changes seen in the evaluation of clinical chemistry and urine parameters, have been shown to be reversible in a previous 13-week rat study of senna. No treatment-related neoplastic changes were observed in any of the examined organs. Based upon these data, it is concluded that senna is not carcinogenic even after daily administration for 2 years at dosages of up to 300 mg/kg/day in Sprague-Dawley rats.

Falls and health status in elderly women following first eye cataract surgery: a randomised controlled trial.

BACKGROUND/AIM: A third of elderly people fall each year. Poor vision is associated with increased risk of falls. The authors aimed to determine if first eye cataract surgery reduces the risk of falling, and to measure associated health gain. METHODS: 306 women aged over 70, with cataract, were randomised to expedited (approximately 4 weeks) or routine (12 months wait) surgery. Falls were ascertained by diary, with follow up every 3 months. Health status was measured after 6 months. RESULTS: Visual function improved in the operated group (corrected binocular acuity improved by 0.25 logMAR units; 8% had acuity worse than 6/12 compared with 37% of controls). Over 12 months of follow up, 76 (49%) operated participants fell at least once, and 28 (18%) fell more than once. 69 (45%) unoperated participants fell at least once, 38 (25%) fell more than once. Rate of falling was reduced by 34% in the operated group (rate ratio 0.66, 95% confidence interval 0.45 to 0.96, p = 0.03). Activity, anxiety, depression, confidence, visual disability, and handicap all improved in the operated group compared with the control group. Four participants in the operated group had fractures (3%), compared with 12 (8%) in the control group (p = 0.04). CONCLUSION: First eye cataract surgery reduces the rate of falling, and risk of fractures and improves visual function and general health status.

A 13-week oral toxicity study of senna in the rat with an 8-week recovery period.

Senna was administered by gavage to Sprague Dawley rats once daily at dose levels of 0, 100, 300, 750 or 1500 mg/kg for up to 13 consecutive weeks followed by an 8-week recovery period for selected animals. Dose- and treatment-related clinical signs included abnormal feces, which were seen to varying degrees from animals at 300 mg/kg per day and more. Animals receiving 750 or 1500 mg/kg per day had significantly reduced body weight gain (males only) and, related to the laxative properties of senna, increased water consumption and notable changes in electrolytes in both serum and urine. At both the terminal and recovery phase necropsy, an increase in absolute and relative kidney weights was seen for male and female animals receiving 750 and/or 1500 mg/kg per day. A dark discoloration of the kidneys was observed at necropsy along with histopathological changes in the kidneys (slight to moderate tubular basophilia and pigment deposits) at 300 mg/kg and above. However, there were no indications in laboratory parameters of any renal dysfunction. In addition, for all treated groups, minimal to slight hyperplasia was recorded in the forestomach and large intestine. Following 8 weeks of recovery, with the exception of the brown pigment in the kidneys, there were no histopathological abnormalities. Thus, the biochemical and morphological changes seen following 13 weeks of treatment of senna significantly reversed following 8 weeks of recovery.

Visual outcome of malignant hypertension in young people.

A retrospective review was carried out of patients under 16 years old with malignant hypertension, who had been referred to a teaching hospital ophthalmology department because of reduced visual acuity. Four patients (three girls, one boy) were seen between 1994 and 2000 with a mean age at presentation of 11.5 years (range 9-15). In the short term, visual acuity improved after control of blood pressure in all four patients. However, in the long term, two patients were registered blind one to two years after presentation, one because of a choroidal neovascular membrane developing at the macula, and the other because of progressive optic neuropathy. Both of these patients had a longer duration of symptoms before diagnosis, worse visual acuity, and higher blood pressure at presentation when compared with the patients who made a good visual recovery. These observations suggest that early diagnosis of malignant hypertension in children is essential in reducing the likelihood of permanent severe visual damage.