Gastrointestinal sequelae and growth impairment at school age following necrotising enterocolitis in the newborn period.

AIM: To evaluate gastrointestinal sequelae and growth impairment at school age in children who suffered from necrotising enterocolitis (NEC). METHODS: This historic cohort study compared all surviving children born in Denmark between 1 January 2002 and 31 December 2011 with NEC in the newborn period, to surviving children without NEC, but same gestational age, birthweight and year of birth. Outcomes were investigated through a parental questionnaire, including gastrointestinal and growth-related outcomes. We performed exploratory ad hoc analysis, by adjusting for possible confounding and by dividing NEC children into surgical and medical. RESULTS: In total, 163 children with NEC (50%) and 237 (36%) without NEC completed the parental questionnaire. Episodes of diarrhoea were more often reported in the NEC group (p = 0.0002). The increased risk seemed to be limited to those who underwent surgery for NEC. The absence from school (1.67 versus 1.31 days), rate of low height for age (17.9 versus 12.1%) and weight (29.9 versus 31.6 kg) did not differ significantly between children with NEC and children without NEC. CONCLUSION: Our findings suggest that long-term gastrointestinal complications following NEC appeared to be of little clinical importance at the population level and therefore do not encourage specific routine follow-up.

Poor validity of the routine diagnosis of necrotising enterocolitis in preterm infants at discharge.

AIM: Necrotising enterocolitis contributes considerably to the mortality of preterm infants, but most questions remain unsolved after decades of extensive research. This Danish study investigated the validity of necrotising enterocolitis diagnoses at discharge according to Bell's staging system. METHODS: We conducted a retrospective single-centre cohort study of 714 preterm infants with a gestational age of less than 30 weeks born in 2006-2013. The infants were diagnosed with necrotising enterocolitis according to Bell's stages 2-3 at discharge and in retrospect by an expert panel, which served as our gold standard. RESULTS: The sensitivity of necrotising enterocolitis diagnosed at discharge was 0.72-0.75 depending on whether spontaneous intestinal perforation was included as necrotising enterocolitis or not. The positive predictive value of the diagnosis was 0.49-0.61. The incidence was significantly higher when diagnosed at discharge than when diagnosed by the expert panel (11.1 versus 9.0%, p = 0.03). The mortality rate for infants who were underdiagnosed at discharge was 50.0%, and it was 25.8% for infants who were overdiagnosed (p = 0.10). CONCLUSION: We found poor validity for the discharge diagnosis of necrotising enterocolitis. In future, a better way of defining the disease is needed for large-scale epidemiologic research.

Surgical findings during exploratory laparotomy are closely related to mortality in premature infants with necrotising enterocolitis.

AIM: This study investigated whether a correlation existed between surgical findings during the first laparotomy for necrotising enterocolitis (NEC) and death and, or, disease progression. METHODS: We included infants admitted within one day of birth to our tertiary neonatal department at Rigshospitalet, Denmark, from 2006 to 2015, who underwent a laparotomy for acute NEC. They were classified according to the locality and extent of intestinal necrosis by a paediatric surgeon, based on the surgical findings. We correlated the surgical findings with postoperative outcomes, namely death and, or, progression of NEC. RESULTS: The first laparotomy showed that 48 infants had NEC, including 21 who demonstrated postoperative progression. Of these, six died before undergoing another laparotomy and 14 of the 15 infants who underwent relaparotomy also died. There was a significant association between surgical findings and NEC-related mortality (p = 0.03). The association between surgical findings and the progression of NEC was also significant (p < 0.0001). CONCLUSION: Surgical findings during laparotomy for NEC were strongly correlated with mortality, which was close to 100% after relaparotomy. Considering the discouraging outcome, further studies should focus on alternative surgical approaches, such as proximal diverting jejunostomy and the clip and drop technique for the treatment of severe NEC.

Add-on perampanel and aggressive behaviour in severe drug-resistant focal epilepsies.

This study aimed to investigate the incidence of aggressiveness in patients with severe drug-refractory focal epilepsy (DRE) who started perampanel (PER) as add-on treatment, and to identify possible predisposing factors. Data on 49 consecutive patients with severe DRE who initiated PER were retrospectively collected. Twelve of the 49 patients experienced aggressiveness as adverse event related to PER treatment, one third of them on low (2-4 mg/day) PER dosages. PER was discontinued in 10/12 patients because of aggressive behaviors. Aggressiveness could appear after several months or even more than one year of PER treatment. One third of patients with PER-related aggressiveness had intellectual disabilities and 5/12 patients took levetiracetam as a concomitant antiepileptic drug. Our study suggests that the occurrence of aggressive behaviors in patients with severe DRE is not uncommon during PER treatment and that it may occur after months or even years of treatment with a stable dosage, requiring PER discontinuation in the great majority of patients.

Perampanel as add-on treatment in refractory focal epilepsy. The Dianalund experience.

BACKGROUND: Perampanel (PER) is an antagonist of AMPA receptors that has been approved for adjunctive treatment of partial-onset seizures. AIMS: To evaluate effectiveness and safety of PER as add-on treatment in patients with severely refractory focal epilepsy. METHODS: PER was introduced as add-on treatment in 22 consecutive patients with drug-resistant focal epilepsy. PER was started with 2 mg/day at bedtime and was up-titrated by 2 mg/day every 2-4 weeks. RESULTS: All patients suffered from severely refractory focal epilepsy (86% took 2 or more AEDs prior PER initiation; 40% had been submitted to surgery or were surgery candidates; 7 had VNS). After 12 months since PER initiation, the retention rate was 54.5% and the responder rate was 27.2%, including 9.1% seizure-free patients. Mean PER dose in the responders was 8 mg/day (range 4-10). Most common side effects were tiredness, behavioral changes (primarily aggressivity), dizziness and were reported in 59.1% of patients, leading to PER discontinuation in 31.8% of subjects. CONCLUSIONS: PER as add-on treatment can achieve clinically meaningful improvement in patients suffering from severely refractory focal epilepses. Further studies are warranted to explore the tolerability profile, with particular focus on psychiatric adverse events.

Bismuth ions are metabolized into autometallographic traceable bismuth-sulphur quantum dots.

Bismuth - sulphur quantum dots can be silver enhanced by autometallography (AMG). In the present study, autometallographic silver enhanced bismuth-sulphur nanocrystals were isolated from unfixed cryo-sections of kidneys and livers of rats exposed to bismuth (Bi207) subnitrate. After being subjected to AMG all the organic material was removed by sonication and enzymatic digestion and the silver enhanced Bi-S quantum dots spun down by an ultracentrifuge and analyzed by scintillation. The analysis showed that the autometallographic technique traces approximately 94% of the total bismuth. This implies that the injected bismuth is ultimately captured in bismuth-sulphur quantum dots, i.e., that Bi-S nanocrystals are the end product of bismuth metabolism.

Atomic force microscope tip spontaneous retraction from dielectric surfaces under applied electrostatic potential.

A time-resolved method for tip' retraction at micros-scale away from dielectric surfaces has been developed. Analysis of the forces in the system comprising AFM tip, water meniscus, and polymer film suggests that an electrostatic repulsion of the tip from the surface in the double-layered (water and polymer) system, and water condensation in the tip-surface junction are the dominant factors enabling the mechanical work for tip retraction. Nanostructures of 5-80 nm height are formed in polymeric surfaces as a result. This interesting physical phenomenon could be used for nanostructures patterning in polymeric materials at enhanced aspect ratio.

Computer-assisted visualization of the rat epididymis: a methodological study based on paraffin sections autometallographically stained for zinc ions.

A concept for the computer-assisted visualization of tubular organs is presented. Unmarked histological zinc-stained serial sections from the epididymis of the Wistar rat were aligned to demonstrate the concept. Virtual images were made through the aligned sections and served as controls for the alignment process. Animation of the serial sections and the virtual images revealed new information about the structure of the organ under investigation. The analysis was used to upgrade the anatomical knowledge of rat epididymis by describing how the epididymal duct runs through the structure. The proximal parts of the epididymis contain large communicating septa of connective tissue dividing the caput and the upper part of the corpus epididymidis into segments. The tortuousness was high in the caput with many turns within a small area of the epididymis, whereas longer loops were found in the lower part of the corpus and cauda epididymidis. The tube of the vas deferens was found to become an integrated part of the ductal system in the cauda epididymidis, although it was histologically easy to distinguish from the epididymal duct. The total number of cross-sections of the ductus epididymidis in the 2254, 15-microm-thick, tissue sections analysed was 104700, giving a minimum length of the ductal system of 1.5 m.

Autometallographic silver enhancement of zinc sulfide crystals created in cryostat sections from human brain biopsies: a new technique that makes it feasible to demonstrate zinc ions in tissue sections from biopsies and early autopsy material.

We present a new technique that allows zinc ions in synaptic and secretory vesicles of biopsy and early autopsy material (< 2 hr post mortem) to be transformed to nanometer-sized zinc sulfide crystal lattices for subsequent autometallographic (AMG) development. Human brain biopsies, or other tissue samples containing zinc-enriched (ZEN) cells, are frozen in liquid nitrogen or by CO2 gas immediately after removal. The tissue blocks are cut in a cryostat and the sections placed on glass slides. The slides are transferred to an H2S exposure chamber placed in a -15 C freezer. After 1-24 hr of gas exposure the sections are removed from the chamber, fixed while thawing, and dehydrated. The sections are then exposed to an AMG developer. AMG causes silver enhancement of zinc sulfide crystal lattices created in the tissues through the H2S exposure, making them visible. It is imperative that the tissues are frozen instantaneously after removal, because loosely bound or free zinc ions start leaving their vesicular compartment soon after death. The AMG technique can, despite inadequate fixation and damage to the tissue caused by freezing, also be used to trace zinc ions at ultrastructural levels, and it is demonstrated that zinc ions in the human neocortex are located in synaptic vesicles. In the few human biopsies analyzed thus far, the light microscopic pattern created by the silver-enhanced ZEN terminals resembles that seen in the neocortex of rat brain. The technique has been applied to cryostat sections from neocortex biopsies of five individuals undergoing brain surgery. Biopsies from three patients resulted in satisfactory AMG-stained sections. Rat brains removed and frozen immediately after decapitation constituted the material on which the present technique was developed. Such material results in an almost uniform high quality of staining, and we found that unexposed sections can be stored for at least 5 months at -80 C without ensuing significant loss of AMG staining intensity.

Autometallographic demonstration of zinc ions in rat sperm cells.

An in-vitro technique for autometallographic (AMG) demonstration of chelatable zinc in electroejaculated sperm cells and spermatozoa from the epididymis is presented and the localization of zinc ions in rat spermatozoa is described. Sperm cells from caput epididymis showed zinc staining in all parts of the tail and a sparse, dispersed staining in the acrosome. Spermatozoa from cauda epididymis showed heavy staining in the acrosome but no staining in the tail, or post-acrosomal part of the sperm head. This distinct acrosomal AMG staining was also found in ejaculated spermatozoa, but additionally a segmentation of the tail was seen based on differences in staining intensity. The membrane penetrating chelator diethyldithiocarbamate (DEDTC) was found to block the AMG staining whereas calcium-EDTA, known not to pass through cell membranes, did not influence the staining, proving that the detected zinc ions are intracellularly located. Two different approaches for demonstrating the presence of a chelatable zinc pool at electron microscope levels are presented, and the ultrastructural presence of AMG grains located in the acrosome and in the mitochondria of the midpiece is demonstrated. It is postulated that an exchange of zinc ions takes place between the epididymal epithelium and the sperm cells as they pass along the epididymal duct.

Increased amount of zinc in the hippocampus and amygdala of Alzheimer's diseased brains: a proton-induced X-ray emission spectroscopic analysis of cryostat sections from autopsy material.

Zinc has been implicated as a contributing cause of the neuropathology of Alzheimer's disease (AD), but consensus on the zinc content of AD brains has not yet been established. In the present study, multi-element PIXE was used to measure zinc in cryostat sections of brain tissue from AD patients and from normal control subjects. Compared to their age-matched controls, the AD patients showed an increase in zinc in the hippocampal and amygdalar regions. The instrumental PIXE assays do not show whether the zinc changes are due to altered zinc in the boutons of Zinc-ENriched (ZEN) neurons, i.e., zinc ions in synaptic vesicles, or to changes in the amount of zinc tightly bound to macromolecules. We hypothesise that the increased zinc level is caused by an increase in the amount of ZEN terminals. Such an increase could be the result of a sprout of ZEN terminals in diseased areas of the brain.

Ultrastructural localization of zinc ions in the rat prostate: an autometallographic study.

BACKGROUND: The prostate contains high amounts of free zinc ions which are excreted into the seminal fluid. The extra- and intracellular distribution of zinc ions using the highly specific autometallographical (AMG) method is described. METHODS: Prostates from sulfide-perfused rats were excised, and the ZnS crystals were silver-enhanced to sizes detectable by the electron and light microscope. RESULTS: AMGZnS grains were found primarily in the acinic lumen of the lateral lobes. The dorsal lobe stained only faintly, while the ventral lobe was void of grains. At ultrastructural levels, the presence of zinc ions was confined to apical secretory vesicles and lysosome-like structure of the epithelium of mainly the lateral lobes. CONCLUSIONS: We suggest a constant secretion of zinc ions from the epithelial cells into both the acinar lumen and the intercellular canaliculi, and that the zinc enriched secretory cells in the prostate belong to a system of glandular cells that uses zinc ions to aggregate macromolecules to be excreted.

Distinct differences in partial oxygen pressure at micrometer ranges in the rat hippocampal region.

A mapping at micrometer ranges of the partial oxygen pressure in the rat hippocampus was performed. The oxygen tension in the rat hippocampal region was measured using a glass oxygen microsensor in 30-microm steps along straight lines at a set of stereotactic coordinates. In the hippocampus the pattern of the oxygen tensions reflected the autometallographic zinc sulphide (AMG(ZnS)) pattern, i.e. the pattern of zinc enriched (ZEN) terminals. The highest levels of oxygen tension were recorded in the areas that are most heavily stained with the autometallographic zinc sulphide (AMG(ZnS)) method, like hilus fasciae dentatae. The zinc ions located in synaptic vesicles of the ZEN terminals can also be demonstrated by AMG silver amplification in brains from animals in vivo treated with sodium selenite. This method depends on the presence of a substantial reduction capacity of the tissues as selenite ions (SeO(2)(3)-) must to be reduced to selenide ions (Se2-) before the catalytic zinc selenide crystals can be formed. At some point, either during the transport from the infusion site to the actual target tissue or in the target tissue itself, selenium is reduced from Se(+ IV) to Se(- II). The importance of the reduction capacity of the target tissue in this process is demonstrated by the fact that areas found to have the highest concentration of zinc ions, e.g. hilus fasciae dentatae and the mossy fibres of CA3, are almost unstained after 1 h of i.p. Na2SeO3 exposure. An explanation of this phenomenon could be that the reduction process Se(+ IV) <==> Se(- II) leading to the formation of Se2- is moved to the left by the presence of oxygen, thus inhibiting the precipitation of ZnSe crystals. It is suggested that the subtle oxygen pressure pattern found in the rat hippocampus might also reflect essential biological zinc-related mechanisms vital to brain function.

PC-assisted three-dimensional description of organs containing tubular structures, applied on the epididymis of the rat.

A concept for three-dimensional computer-assisted reconstruction of tubular organs, e.g. the epididymis, is described. Histologic serial sections without artificial landmarks from the epididymis of the Wistar rat were aligned. Virtual images through the aligned sections served as a control of the alignment process and can reveal new information about the structure of the organ under investigation. The method can be used for improving the anatomical description of the epididymis, i.e. how the ductus epididymidis is coiled along this organ. Other tubular tissues and organs can be investigated and analysed with this PC-assisted method, e.g. testis and kidney.

Histochemical demonstration of zinc ions in human epididymis using autometallography.

A recently described autometallographic technique, allowing demonstration of chelatable zinc in human biopsy material, was applied to cryostat sections from biopsies of human epididymis. Sections from the rat epididymis were used as control materials to examine the quality of the method compared with a previously used autometallographic method. The human epididymis exhibits heavy staining in the head of the epididymis and only small amounts of zinc in the body and tail of the organ. The zinc staining was found in the apical part of the ciliated cells and in the lumen. The present technique can be used to localize zinc ions at ultrastructural levels. Zinc grains were localized in lysosome-like bodies and secretory granules of the ciliated cells. The luminal staining was present as free, evenly dispersed zinc grains or attached to sperm cells and stereocilia in the lumen. The large differences in staining patterns along the epididymal tract in humans and rats suggest that zinc ions are important for the maturation of sperm cells.

How to detect gold, silver and mercury in human brain and other tissues by autometallographic silver amplification.

Gold, silver, mercury and zinc bind chemically to sulphide or selenide ions and create crystal lattices that can be detected in histological sections by a silver amplification technique called autometallography (AMG). The technique specifically magnifies such nanometer-sized catalytic crystals. For each metal, a detailed protocol has been worked out. If several different AMG metals/metal molecules are present in the same tissue, it is possible to distinguish one from another. The AMG technique is based on the capability of small crystal lattices of the aforementioned metals and metal molecules to initiate AMG silver amplification. Electrons released from adhering hydroquinone molecules reduce silver ions that are integrally connected with the crystal lattices. In this manner, particles consisting of only a few atoms of, say, gold, or molecules of mercury selenide (Figure 1), can be silver amplified to a size at which they can be detected in the electron microscope, or even further to dimensions that can be observed in the light microscope. Thus the AMG technique opens up the possibility of visualizing gold, e.g. in the nervous system of rheumatic patients who have been treated with aurothiomalate. Mercury can similarly be visualized in tissues from individuals who have been exposed to mercury, either through leaching from amalgam dental fillings, through eating fish, or by occupational exposure, and silver in the central (CNS) and peripheral nervous systems (PNS) and other tissues from individuals exposed to silver in one form or another. In the future, the possibility of demonstrating vesicular zinc, a particular pool of endogenous zinc that is found in terminals of zinc-enriched neurons (ZEN neurons), might prove valuable for pathological interpretation of diseases such as Alzheimer's disease. The vesicular zinc, present in some of the synaptic vesicles of ZEN neuron terminals, is most impressively demonstrated by AMG in telencephalic structures. It is becoming increasingly indisputable that vesicular zinc is related to synaptic activity influencing or modulating facilitatory synapses. ZEN neurons are probably a sub-population of glutaminergic neurons. A technique for the post-mortem demonstration of vesicular zinc in terminals of ZEN neurons in human brains is therefore urgently required.

Autometallographic detection of silver in hypothalamic neurons of rats exposed to silver nitrate.

The silver amplification technique, autometallography, has been used to reveal silver accumulation in neurons in the hypothalamus of male Wistar-Kyoto rats. Silver penetrated the blood-brain barrier after exposure to silver nitrate, and differences in staining intensity were found between the hypothalamic nuclei. When using light microscopy, the silver staining of the hypothalamic neurons was highly heterogeneous. Silver was detected exclusively in lysosomes of the loaded neurons. Aspects of this heterogeneous localization are discussed in relation to the distribution of autometallographically developed gold and mercury.

Efficiency of autometallographic detection of mercury in the rat kidney.

The autometallographic silver enhancement method is a method for subcellular localization of some heavy metals, such as mercury. However, no quantitative estimate has been made of the amount of mercury demonstrated by the method. In this study, pellets of autometallographic silver grains were prepared from unfixed kidney slices of rats exposed i.p. to mercury chloride containing trace amounts of 203Hg. The slices were silver-enhanced, and subsequently all organic material was removed by enzymatic digestion. During all stages of the experiment the solutions and tissue were gamma-counted. The analysis showed that the final pellets contained approximately 30% of the mercury compared to that found in the slices prior to development and that the mercury was probably located in lysosomes.