Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy.

Determining how neurons transform synaptic input and encode information in action potential (AP) firing output is required for understanding dendritic integration, neural transforms and encoding. Limitations in the speed of imaging 3D volumes of brain encompassing complex dendritic arbors in vivo using conventional galvanometer mirror-based laser-scanning microscopy has hampered fully capturing fluorescent sensors of activity throughout an individual neuron's entire complement of synaptic inputs and somatic APs. To address this problem, we have developed a two-photon microscope that achieves high-speed scanning by employing inertia-free acousto-optic deflectors (AODs) for laser beam positioning, enabling random-access sampling of hundreds to thousands of points-of-interest restricted to a predetermined neuronal structure, avoiding wasted scanning of surrounding extracellular tissue. This system is capable of comprehensive imaging of the activity of single neurons within the intact and awake vertebrate brain. Here, we demonstrate imaging of tectal neurons within the brains of albino Xenopus laevis tadpoles labeled using single-cell electroporation for expression of a red space-filling fluorophore to determine dendritic arbor morphology, and either the calcium sensor jGCaMP7s or the glutamate sensor iGluSnFR as indicators of neural activity. Using discrete, point-of-interest scanning we achieve sampling rates of 3 Hz for saturation sampling of entire arbors at 2 µm resolution, 6 Hz for sequentially sampling 3 volumes encompassing the dendritic arbor and soma, and 200-250 Hz for scanning individual planes through the dendritic arbor. This system allows investigations of sensory-evoked information input-output relationships of neurons within the intact and awake brain.

Automating Event-detection of Brain Neuron Synaptic Activity and Action Potential Firing in vivo using a Random-access Multiphoton Laser Scanning Microscope for Real-time Analysis.

Determining how a neuron computes requires an understanding of the complex spatiotemporal relationship between its input (e.g. synaptic input as a result of external stimuli) and action potential output. Recent advances in in vivo, laser-scanning multiphoton technology, known as random-access microscopy (RAM), can capture this relationship by imaging fluorescent light, emitted from calcium-sensitive biosensors responding to synaptic and action potential firing in a neuron's full dendritic arbor and cell body. Ideally, a continuous output of fluorescent intensities from the neuron would be converted to a binary output (`event', 'or no-event'). These binary events can be used to correlate temporal and spatial associations between the input and output. However, neurons contain hundreds-to-thousands of synapses on the dendritic arbors generating an enormous quantity of data composed of physiological signals, which vary greatly in shape and size. Thus, automating data-processing tasks is essential to support high-throughput analysis for real-time/post-processing operations and to improve operators' comprehension of the data used to decipher neuron computations. Here, we describe an automated software algorithm to detect brain neuron events in real-time using an acousto-optic, multiphoton, laser scanning RAM developed in our laboratory. The fluorescent light intensities, from a genetically encoded, calcium biosensor (GCAMP 6m), are measured by our RAM system and are input to our 'event-detector', which converts them to a binary output meant for real-time applications. We evaluate three algorithms for this purpose: exponentially weighted moving average, cumulative sum, and template matching; present each algorithm's performance; and discuss user-feasibility of each. We validated our system in vivo, using the visual circuit of the Xenopus laevis.

Diagnosing a learning practice: the validity and reliability of a learning practice inventory.

BACKGROUND: There is an increasing literature on learning organisations as a way of fostering communication, teamwork, collaboration and collective learning, thereby promoting quality improvement and enhancing patient safety. An increasing number of instruments are being developed in an attempt to measure learning organisation characteristics. However, the majority of these tools are created for a business setting, have not been scientifically tested and have not been applied in healthcare. OBJECTIVE: To evaluate elements of the validity and reliability of an instrument (ie, learning practice inventory (LPI)) for diagnosing learning practice characteristics in primary healthcare. METHOD: Content validity was evaluated using a modified nominal group technique and a content validity rating scale. Construct validity and reliability evaluation was undertaken with 10 staff members from 10 general practices in the west of Scotland. Staff completed the inventory twice, 4-6 weeks apart. Applying generalisability theory, a variance component analysis was performed. RESULTS: The main findings present evidence that the inventory has acceptable reliability and content validity. The results also demonstrate that the inventory can reflect the consistent and uniquely different perspectives of particular designations of staff within a practice. It is possible to compare practices' overall learning environments and to identify specific areas of practice strength as well as areas for development. CONCLUSION: This study demonstrates the psychometric properties of a learning practice diagnostic inventory. It highlights the consistently different perspectives that individual staff groups have on the function of their practice, suggesting that the success of quality improvement initiatives may be compromised without the involvement and true engagement of each staff group.

Visualizing CD4 T-cell migration into inflamed skin and its inhibition by CCR4/CCR10 blockades using in vivo imaging model.

BACKGROUND: Chemokines are critical mediators of T-cell homing into inflamed skin. The complex nature of this multicellular response makes it difficult to analyse mechanisms mediating the early responses in vivo. OBJECTIVES: To visualize directly T-cell homing into inflamed skin and its inhibition by blockades using a unique noninvasive confocal microscopy. MATERIALS AND METHODS: A mouse model of allergic contact dermatitis was used. T cells from oxazolone-sensitized and -challenged Balb/c mice were first analysed phenotypically in vitro. CD4 T cells were then labelled with a tracker dye and transferred into Balb/c-SCID mice. The recipient mice were challenged with oxazolone and CD4 T-cell homing into inflamed skin was visualized. RESULTS: T cells with the skin homing receptors CCR4 and CCR10 were increased in the affected skin and draining lymph nodes, and effectively attracted by their specific chemokines CCL17, CCL22 and CCL27 in vitro. Using in vivo imaging, T-cell migration into the inflamed skin was observed at 2 h after application, peaking at 12 h and continuing for 48 h. Simultaneous systemic administration of neutralizing antibodies against CCR4 ligands (CCL17 and CCL22) and CCR10 ligand (CCL27) led to a significant suppression of T-cell migration and skin inflammation. CONCLUSIONS: Our data indicate that these tissue-selective adhesion molecules and chemokine/receptor pathways act in concert to attract specialized T-cell populations to mediate cutaneous inflammation. The in vivo imaging technique can be applicable to other models of cutaneous diseases to help with better understanding of the pathogenesis and monitoring the therapeutic effects.

A new genetic disorder in mitochondrial fatty acid beta-oxidation: ACAD9 deficiency.

The acyl-CoA dehydrogenases are a family of multimeric flavoenzymes that catalyze the alpha,beta -dehydrogenation of acyl-CoA esters in fatty acid beta -oxidation and amino acid catabolism. Genetic defects have been identified in most of the acyl-CoA dehydrogenases in humans. Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified acyl-CoA dehydrogenase that demonstrates maximum activity with unsaturated long-chain acyl-CoAs. We now report three cases of ACAD9 deficiency. Patient 1 was a 14-year-old, previously healthy boy who died of a Reye-like episode and cerebellar stroke triggered by a mild viral illness and ingestion of aspirin. Patient 2 was a 10-year-old girl who first presented at age 4 mo with recurrent episodes of acute liver dysfunction and hypoglycemia, with otherwise minor illnesses. Patient 3 was a 4.5-year-old girl who died of cardiomyopathy and whose sibling also died of cardiomyopathy at age 21 mo. Mild chronic neurologic dysfunction was reported in all three patients. Defects in ACAD9 mRNA were identified in the first two patients, and all patients manifested marked defects in ACAD9 protein. Despite a significant overlap of substrate specificity, it appears that ACAD9 and very-long-chain acyl-CoA dehydrogenase are unable to compensate for each other in patients with either deficiency. Studies of the tissue distribution and gene regulation of ACAD9 and very-long-chain acyl-CoA dehydrogenase identify the presence of two independently regulated functional pathways for long-chain fat metabolism, indicating that these two enzymes are likely to be involved in different physiological functions.

An unreported variation of the cervical vagus nerve: anatomical and histological observations.

Variations involving the cervical portion of the vagus nerve are seemingly very rare. We report an adult male found to harbour a right cervical vagus nerve that crossed anterior to the right common carotid artery to terminate in the lateral aspect of the thyroid gland. A very small continuation of this nerve was found to continue distally into the thorax. Histologically, this part of the vagus nerve did not contain ganglion or other cell bodies. There were no heterologous inclusions (thyroid, parathyroid, thymus, salivary gland or branchial cleft remnants) present. Although grossly there was a connection into the thyroid gland, this was not observed histologically. No signs of trauma were found to the ipsilateral neck region. We hypothesise that this variation is due to entanglement between the thyroid gland and cervical vagus nerve during development. This rare variation might be considered by the clinician who operates in the cervical region or interprets imaging of the neck. To our knowledge, a vagus nerve with the above described morphology has not been described.

Synchronous bifocal alveolar rhabdomyosarcoma: a case report.

We report a case of bifocal rhabdomyosarcoma involving the hand and thigh in an 11-year-old female. We highlight the importance of a thorough clinical examination and an aggressive surgical approach in which each lesion is treated as a separate primary.

Dwarfism, occult spinal dysraphism, and presacral myxopapillary ependymoma with an epidermoid cyst in a child.

The authors present a case of a child with dwarfism that was noted to be developmentally delayed. Imaging revealed atlantoaxial instability, occult spinal dysraphism, and a presacral mass. Histopathology of the presacral lesion was that of a myxopapillary ependymoma with epidermoid cyst. We believe this to be the first report in the extant medical literature of this constellation of findings in the same patient. However, there are rare reports indicating a possible association of occult spinal dysraphism and the simultaneous occurrence of spinal ependymomas. Further case reports are necessary to discern whether these pathological entities are true low rate associations that the clinician should consider in their evaluation of these patients.

Histological appearance of a chronically stimulated vagus nerve in a pediatric patient.

Histological analysis of chronically stimulated human vagus nerves is lacking in the literature. In this study, we describe the first microscopic findings in a chronically stimulated left vagus nerve from a pediatric patient. Our results show many histological changes in and around the stimulated nerve with severe demyelination. Further long-term clinical and postmortem examinations of chronically stimulated vagus nerves in both children and adults are needed to ascertain whether prolonged exposure to electrical current can cause clinical dysfunction of this nerve.

Evaluation of a new percutaneous dilatational tracheostomy set apparatus.

Percutaneous tracheostomy is a well established technique used primarily to assist weaning from mechanical ventilation on many intensive care units. We report our experiences of a total of 36 procedures performed with the new Blue Rhino Percutaneous Tracheostomy Introducer Set developed by Ciaglia. The technique was successful in all cases and was simpler and quicker to perform than with the earlier Ciaglia percutaneous tracheostomy set. Difficulties were encountered when using Shiley tracheostomy tubes. Significant complications included one posterior wall tear and one tracheal cartilage ring fracture.

Molecular mechanisms contributing to necrotizing enterocolitis.

OBJECTIVE: To examine the cellular mechanisms involved in the pathogenesis of necrotizing enterocolitis (NEC). SUMMARY BACKGROUND DATA: Necrotizing enterocolitis is a major cause of death and complications in neonates; the cellular mechanisms responsible for NEC are unknown. The inducible form of cyclooxygenase (i.e., COX-2) is activated by the transcription factor nuclear factor (NF)-kappaB and is thought to play a role in inflammation. METHODS: Segments of perforated and adjacent uninvolved small intestine from neonates with NEC were analyzed for COX-2 expression by immunohistochemistry. NEC was induced in weanling (18 days old) rats by occlusion of superior mesenteric vessels for 1 hour and intraluminal injection of platelet activating factor (50 micro/kg). Small intestine was harvested for protein extraction. Western immunoblot was performed to determine expression of COX-2. Gel shift assays were performed to assess NF-kappaB binding activity. RESULTS: Immunohistochemical analysis showed increased COX-2 protein expression in the perforated intestinal sections of all 36 neonates but not in adjacent normal intestine. Increased expression of COX-2 protein and NF-kappaB binding activity was noted in the small intestine of weanling rats at 0 and 3 hours after induction of NEC. CONCLUSIONS: Increased COX-2 expression was identified in all neonatal intestinal segments resected for perforated NEC. In addition, a coordinate induction of COX-2 expression and NF-kappaB binding was noted in a rodent model of NEC. These findings suggest that the COX-2/NF-kappaB pathway may play a role in the pathogenesis of NEC. Therapeutic agents that target this pathway may prove useful in the treatment or possible prevention of NEC.

Pediatric inflammatory bladder tumors: myofibroblastic and eosinophilic subtypes.

PURPOSE: Benign bladder tumors are rare in children. A number of descriptive terms have previously been used to describe inflammatory tumors with myofibroblastic proliferation or eosinophilic infiltration. We present our experience with these tumors and review the literature. MATERIALS AND METHODS: We retrospectively reviewed the records of all children presenting with a benign focal bladder mass during the last 5 years, including 2 girls and 3 boys 2 to 12 years old (mean age 7). RESULTS: Presenting symptomatology included irritative voiding symptoms, suprapubic pain and hematuria. All bladder masses were diagnosed by ultrasonography and all patients underwent transurethral resection. Three children who had an inflammatory bladder tumor with myofibroblastic features required open tumor excision with preservation of the bladder. In the 2 remaining children urine culture was positive preoperatively and the diagnosis was an inflammatory bladder tumor with eosinophilic infiltration. Transurethral resection of the mass was performed without partial cystectomy. CONCLUSIONS: Inflammatory myofibroblastic and eosinophilic tumors represent forms of focal cystitis with a tumefactive component. Differentiation from the malignant bladder neoplasms of childhood has important consequences for therapy. Management of these benign but aggressive inflammatory lesions involves local control by endoscopic or open surgery with bladder preservation. We encourage refined use of the terminology for inflammatory myofibroblastic and eosinophilic tumors.

Urinalysis: current status and prospects for the future.

More than 300 million routine clinical analyses are performed annually in the United States. Methods for routine clinical urine examination, including detection of bacteriuria, are briefly reviewed. Prospects of some newer, better techniques to carry out such analyses are introduced. A preliminary report is presented on the use of supravital microscopic fluorescence technique (SMFT), employing acridine orange as a non-specific staining fluorochrome. Results of examining 218 unspun urine specimens by SMFT are compared to traditional bacteriologic culture at a large pediatric hospital reference laboratory.

The development and evaluation of a personal learning log for senior house officers.

OBJECT: To develop and evaluate the effect of having a personal learning log on Senior House Officers knowledge and confidence. METHODS: A multiple choice paper and a confidence checklist for two hospital specialties were developed to assess knowledge and confidence. These were administered to a control group and to an intervention group who had the learning log. Both groups completed an evaluation at the end of the post. SETTING: The study took place in Accident & Emergency and Obstetric & Gynaecology posts in Greater Glasgow and Lanarkshire. SUBJECTS: 79 Senior House Officers in Accident & Emergency and 78 Senior House Officers in Obstetrics & Gynaecology. RESULTS: The mean scores in the MCQ and the mode in the confidence checklist increased significantly in both specialties during the post, but there was no significant difference between the control and intervention groups. Forty two learning logs were returned at the end of the study and analysis of these revealed that there was great scope for learning but few documented the specific learning achieved. Evaluation of the posts revealed that some improvements had taken place in teaching and assessment frequency, however, there was scope for further improvement. CONCLUSION: While the problems of hospital training are well documented, an attempt to improve the situation using a learning log did not have a statistically significant impact on SHO knowledge or confidence. A six-month hospital post appears to present many opportunities for learning but these are not exploited. It is suggested that three things are needed. Firstly, active participation by and personalized feedback from a senior member of staff, with training where needed. Secondly, protected time for tutorials with a planned system of formative assessment, and thirdly, a more positive approach to learning by both SHOs and consultants. Once this occurs, a learning log may have a more significant impact on training.

Gastrointestinal autonomic nerve tumors in the pediatric population: a report of four cases and a review of the literature.

BACKGROUND: Gastrointestinal autonomic nerve tumors (GANTs) are a subpopulation of gastrointestinal stromal tumors (GISTs) that are characterized by ultrastructural features resembling enteric autonomic nerve cells, without epithelial, Schwannian, or smooth muscle differentiation. Delineation of the clinicopathologic features of GANT in the pediatric population is lacking. METHODS: The clinicopathologic and outcome data for four pediatric patients with GANT are presented. The data from these patients and four previously reported pediatric patients are summarized and compared with data for GANT in adults. RESULTS: All four cases occurred in females at a mean age of 12.5 years. The primary tumor site was the stomach in all cases, and the mean tumor size was 6.3 cm. Immunocytochemical and ultrastructural examination were essential in distinguishing GANT from GIST in all cases by identifying features of neural origin (neuron specific enolase in all four cases, NFP in three cases, S-100 in two cases, dense core neurosecretory granules in all four cases, and neuritelike processes in all four cases), while failing to identify features of myogenic origin (no desmin, smooth muscle actin, myofilaments, or dense bodies were found in any of the cases). Primary treatment was surgical, with chemotherapy administered to 1 patient at the time of recurrence. All patients are alive after a mean follow-up of 60 months (range, 8 months to 9 years). CONCLUSIONS: Similarities of pediatric GANT to GANT in adults include the need for a high index of suspicion for diagnosis; comparable histopathologic, immunohistochemical, and ultrastructural features; and surgery as the primary therapy. Distinguishing features in children may be a prevalence among females in the second decade, a predominance of smaller gastric tumors, and a positive prognostic value of younger age.

Isolated epithelial cysts of the uvula in children: a report of three cases.

Epidermoid cysts are unusual benign cysts that occur in the oral cavity less than 2% of the time. Epidermoid cysts isolated to the uvula are rare. A review of the literature has revealed only three confirmed cases of isolated uvular epidermoid cysts. We present a series of three such cases, two of which presented with significant symptomatology. The diagnosis and management of these unusual lesions will be discussed, as well as the differential diagnosis.

Extensive tyrosine nitration in human myocardial inflammation: evidence for the presence of peroxynitrite.

OBJECTIVES: Production of nitric oxide via the cytokine-mediated activation of myocardial inducible nitric oxide synthase decreases myocardial contractility. Whether myocardial dysfunction is mediated directly by nitric oxide or indirectly through the formation of secondary reaction products, such as peroxynitrite, has not been established. Peroxynitrite, but not nitric oxide, reacts with the phenolic ring of tyrosine to form the stable product 3-nitro-L-tyrosine. Demonstration of tissue nitrotyrosine residues, therefore, infers the presence of peroxynitrite or related nitrogen-centered oxidants. DESIGN: Retrospective analysis of human autopsy specimens. SETTING: University pathology and basic science laboratories. PATIENTS: Formalin-fixed, paraffin-embedded myocardial tissue samples were obtained from 11 patients with a diagnosis of sepsis, seven patients with a diagnosis of viral myocarditis, and five control patients without clinical or pathologic cardiac disease. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Specific antibodies to nitrotyrosine were utilized to detect nitrotyrosine residues in human autopsy specimens. Cardiac tissue obtained from patients with myocarditis or sepsis demonstrated intense nitrotyrosine immunoreactivity in the endocardium, myocardium, and coronary vascular endothelium and smooth muscle. In contrast, connective tissue elements were without appreciable immunohistochemical staining. Nitrotyrosine antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin, but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. In situ reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. Densitometric analysis of nitrotyrosine immunoreactivity demonstrated significantly higher values for specimens from myocarditis and sepsis patients when compared with control tissue specimens. CONCLUSION: These results demonstrate the formation of peroxynitrite within the myocardium during inflammatory disease states, suggesting a role for peroxynitrite in inflammation-associated myocardial dysfunction.