Apparatus to investigate the insulation impedance and accelerated life-testing of neural interfaces.

OBJECTIVE: Neural interfaces and other implantable micro-devices that use polymer-encapsulated integrated circuits will only be allowed in medical devices when their lifetimes can be estimated from experimental data. An apparatus has been developed and tested that allows hundreds of insulated samples (interdigitated combs) to be aged under accelerated conditions of high temperature and voltage stress. Occasionally, aging is paused while the sample's impedance is measured; the impedance spectrogram may show degradation as it progresses before failure. APPROACH: The design was based on practical considerations which are reviewed. A Solartron Modulab provides the frequency response analyser and the femtoammeter. The apparatus can accommodate batches of samples at several temperatures and with different aging voltage waveforms. It is important to understand features of the spectra that are not due to comb-comb leakage, but come from other places (for example substrate-solution leakage); some have been observed and investigated using SPICE. MAIN RESULTS: The design is described in detail and test results show that it is capable of making measurements over long periods, at least up to 67 °C. Despite the size of the apparatus, background capacitance is about 1 pF and comb-comb capacitances of about 30 pF can be measured down to 10 mHz, an impedance of about 100 GΩ. An important discovery was the advantage of grounding the bathing solution, primarily in that it raises the measurement ceiling. Observation and SPICE simulation shows that leakage from the substrate to the bathing solution can give phase lags >90°, in contrast to comb-comb leakage which reduces phase lag to <90°. SIGNIFICANCE: The value of this paper is that it will facilitate research into the endurance of small implanted devices because, given a description of a proven apparatus, researchers can start building their own apparatus relatively quickly and with confidence.

Development of the right ventricular inflow tract and moderator band: a possible morphological and functional explanation for Mahaim tachycardia.

Atriofascicular accessory bundles with AV-node like conduction properties can sustain atrioventricular (AV) re-entrant tachycardia (Mahaim tachycardia). During early embryogenesis, the AV canal is situated above the primitive left ventricle (LV), and a right AV connection has not been achieved yet. We studied the formation of the right ventricular (RV) inflow tract in relation to the developing cardiac conduction system and hypothesized a morphological explanation for functional atriofascicular bypass tracts. Analysis of lacZ-expression during sequential stages of cardiogenesis was performed in CCS-lacZ transgenic mice (E9.5 to 15.5). Embryos were stained for beta-galactosidase activity and the myocardial marker HHF35. At early stages CCS-lacZ expression was observed in a ring surrounding the AV canal, which connected at the inner curvature to the primary fold. The first sign of formation of the (CCS-lacZ negative) RV inlet component was a groove in the CCS-lacZ positive tissue of the primary fold. Outgrowth of the RV inlet tract resulted in division of the primary fold in a septal part, the trabecula septomarginalis and a lateral part, the moderator band, which extended laterally up to the right AV ring. Electrophysiological measurements in embryonic hearts (E15.5) in which the right atrium (RA) and RV were isolated from the left atrium (LA) and LV supported the functionality of this AV-connection via the moderator band, by demonstrating sequential atrial and ventricular activation in both RA/RV and LA/LV preparations. In conclusion, our observations may provide a possible morphological and functional explanation for atriofascicular accessory pathways via the moderator band, underlying Mahaim tachycardia.

[A boy with a white striped knee on a radiograph]. / Witte strepen op een röntgenfoto van de knie.

A 10-year-old boy presented at the radiology department with pain in the right knee. Radiographs of the knee revealed dens metaphyseal bands and subchondral epiphyseal sclerosis as a result of periodic bisphosphonate administration for the treatment of Legg-Calvé-Perthes disease three years ago.

A novel flexible capacitive load sensor for use in a mobile unicompartmental knee replacement bearing: An in vitro proof of concept study.

Instrumented knee replacements can provide in vivo data quantifying physiological loads acting on the knee. To date instrumented mobile unicompartmental knee replacements (UKR) have not been realised. Ideally instrumentation would be embedded within the polyethylene bearing. This study investigated the feasibility of an embedded flexible capacitive load sensor. A novel flexible capacitive load sensor was developed which could be incorporated into standard manufacturing of compression moulded polyethylene bearings. Dynamic experiments were performed to determine the characteristics of the sensor on a uniaxial servo-hydraulic material testing machine. The instrumented bearing was measured at sinusoidal frequencies between 0.1 and 10Hz, allowing for measurement of typical gait load magnitudes and frequencies. These correspond to frequencies of interest in physiological loading. The loads that were applied were a static load of 390N, corresponding to an equivalent body weight load for UKR, and a dynamic load of ±293N. The frequency transfer response of the sensor suggests a low pass filter response with a -3dB frequency of 10Hz. The proposed embedded capacitive load sensor was shown to be applicable for measuring in vivo loads within a polyethylene mobile UKR bearing.

Epicardial outgrowth inhibition leads to compensatory mesothelial outflow tract collar and abnormal cardiac septation and coronary formation.

In the present study, we investigated the modulatory role of the epicardium in myocardial and coronary development. Epicardial cell tracing experiments have shown that epicardium-derived cells are the source of interstitial myocardial fibroblasts, cushion mesenchyme, and smooth muscle cells. Epicardial outgrowth inhibition studies show abnormalities of the compact myocardial layer, myocardialization of cushion tissue, looping, septation, and coronary vascular formation. Lack of epicardial spreading is partly compensated by mesothelial outgrowth over the conotruncal region. Heterospecific epicardial transplant is able to partially rescue the myocardial development, as well as septation and coronary formation.

Histologic studies on normal and persistent ductus arteriosus in the dog.

The process of anatomic closure of the ductus arteriosus was studied at the ultrastructural level in 15 normal beagles (age 0 hour to 13 days) and in 18 specimens from a strain of dogs with hereditary persistent ductus arteriosus (age 4 hours to 27 days). Normal ductal closure takes place from the pulmonary artery to the aortic end. It is accompanied by a series of histologic changes: 1) separation of the endothelial cells from the internal elastic lamina resulting in a wide region of subendothelial edema; 2) ingrowth and infolding of endothelial cells and migration of undifferentiated smooth muscle cells from the inner media into the subendothelial region; 3) apposition of endothelial cells bordering the lumen; and 4) degenerative changes. In persistent ductus arteriosus, these changes do not occur. The endothelial cells remain closely adhered to the internal elastic lamina and the underlying media is abnormal in structure. In the case of partial persistent ductus arteriosus (ductus diverticulum), both the normal and the abnormal type of wall are found in a single ductus arteriosus. The histologic features of the normal and the persistent ductus arteriosus in the dog resemble those of the normal and the persistent ductus arteriosus in humans, suggesting a similar pathogenesis.

Differences in development of coronary arteries and veins.

OBJECTIVE: The differentiation of the coronary vasculature was studied to establish in particular the formation of the coronary venous system. METHODS: Antibody markers were used to demonstrate endothelial, smooth muscle, and fibroblastic cells in serial sections of embryonic quail hearts. The anti-beta myosin heavy chain and the neuronal marker HNK-1 were added to our incubation protocol. RESULTS: In HH32, the coronary vascular network has developed into a circulatory system with connections to the sinus venosus, the aorta and the right atrium. The connections between the aorta and the right atrium allow for direct arteriovenous shunting. Subsequently, differentiation into coronary arteries and veins occurs with an interposed capillary network. The smooth muscle cells of the coronary arterial media derive from the subepicardial layer, whereas the subepicardially located cardiac veins recrute atrial myocardium, as these cells express the beta-myosin heavy chain antigen. Ganglia are located in the subepicardium close to the vessels, while nerve fibres tend to colocalize with the formed vessel channels. CONCLUSIONS: A new finding is presented in which the subepicardial coronary veins have a media that consists of myocardial cells. The close positional relationship of neural tissue and coronary vessels that penetrate the heart wall is explained as inductive for vessel wall differentiation, but not for invasion into the heart.

Molecular analysis of an extended family with type IA (tyrosinase-negative) oculocutaneous albinism.

We have analyzed the tyrosinase coding region of three individuals having Type IA OCA within an extended family using genomic DNA amplification and dideoxy sequencing. Two of the affected individuals are dizygotic twins. All three have a common missense mutation at codon 81 (Pro----Leu) within exon I. The twins have a second missense mutation at codon 371 (Asn----Thr) within exon III and the third individual has a second missense mutation at codon 47 (Gly----Asp) within exon I. For each of these three individuals, the loss of enzyme function is the result of two different mutations, showing that they are compound heterozygotes of two mutant tyrosinase alleles.

Role of interleukin-6 and transforming growth factor-beta in anorexia nervosa.

Anorexia nervosa is a serious eating disorder characterized by extreme weight loss and abnormalities of the neuroendocrine and immune systems. To determine the potential role of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-beta) in anorexia nervosa, serum concentrations of these cytokines were measured in patients with anorexia nervosa during starvation and after weight gain. Serum IL-6 and TGF-beta concentrations were both significantly elevated during starvation and returned to levels comparable to those of normal-weight controls by the end of therapy. In contrast, serum TNF-alpha levels were undetectable in all patients and controls. Cytokines may play previously unsuspected roles in anorexia nervosa and its complications.

Rostro-caudal polarity in the avian somite related to paraxial segmentation. A study on HNK-1, tenascin and neurofilament expression.

Segmental organization of the vertebrate body is one of the major patterns arising during embryonic development. Somites that play an important role in this process show intrinsic patterns of gene expression and differentiation. The somites become polarized in all three dimensions, rostrocaudal, mediolateral and dorsoventral, the quadrants giving rise to several tissue components. The timing of polarization was studied by means of antibodies against HNK-1, tenascin and neurofilament. Whole mounts and serial sections of quail and chick embryos show that somites are already polarized at the moment of their segregation from the segmental plate. The rostral hemisomite carries the HNK-1 epitope preferentially, while the caudal hemisomite stains more strongly for tenascin. HNK-1-stained areas in the segmental plate strongly relate to the notochordal sheath, suggesting that axial structures determine the fate of paraxial structures. Neural crest cells were only seen to colonize the rostral part of a somite after they had differentiated into HNK-1 positive cells. Their colonization pattern seems to be guided by the segmental organization of the somite. Moreover, this somite organization probably dictates the organization of both sensory and motor fibres converging towards the segmental dorsal root ganglia, justifying a shift in the connections between neural tube and somites. This segmental shift takes place over one quarter of a somite length in a rostral direction.

Axial rotation of murine embryos, a study of asymmetric mitotic activity in the neural tube of somite stages.

Axial rotation is an important event during a certain period of development of Amniote embryos. In murine embryos a sharp lordosis changes into a kyphosis. The result is the typical fetal position. In this study a temporal and topological relation is found between an asymmetric mitotic activity in the neural tube and the rotation process. The mitotic asymmetry is lost when rotation is completed. A causal relationship between mitotic activity and rotation is postulated.

A SEM study on the development of the ventricular surface morphology in the diencephalon of the rat.

The morphogenesis of the ventricular surface of the diencephalon of the rat was studied using scanning electron microscopy, cryostat serial sections and direct observations under a dissection microscope. Based on these observations a description is given of the neuromeres present within the prosencephalon and of the termination of the sulcus limitans. Two conclusions are reached. First, three neuromeres are present in the prosencephalon. Neuromere I consists of the telencephalon, the hypothalamic regions and the parencephalon anterius. Neuromere II is the parencephalon posterius, neuromere III the synencephalon. Second, the sulcus limitans terminates ventrally in the parencephalon posterius and does not continue towards the preoptic recess. No exact termination point of the sulcus limitans could be delineated.

Cytokeratins as a marker for epicardial formation in the quail embryo.

Several techniques have been used to visualize the migration pattern of the epicardial cells from the proepicardial organ over the myocardial surface. As the epicardial cells contain keratin tonofilament bundles, we have incubated 92 whole-mount quail hearts with an anti-keratin antibody. This immunohistochemical method showed that the complete epicardial covering of the embryonic heart is preceded by the formation of three epicardial rings. The epicardial rings are formed on the outer myocardial surface in the grooves that separate the cardiac segments from each other. We have also documented timing and patterning of isolated epicardial islands. They are not encountered at random over the myocardial surface, but only along the edge of the advancing epicardial front border and in two defined future epicardial ring areas on the ventral side of the outflow tract. The epicardial islands suggest that in the quail free-floating parts of epicardium can attach to the myocardium. Characteristics of the surface of the myocardium at the transitional zones between the cardiac segments, as well as the three-dimensional remodelling of the heart during cardiac morphogenesis seem to play a role in the pattern in which the epicardium eventually completely ensheaths the myocardial surface. Congenital heart defects are often related to malpositioned transitional zones that dictate the pattern of epicardial outgrowth. As the embryonic position of the epicardial rings is mirrored in the pattern of the main arterial stems, the coronary vascularization pattern might be altered in congenitally malformed hearts as well.

Vascular endothelial cells migrate centripetally within embryonic arteries.

Migration of vascular endothelial cells was traced in quail-chick chimeras. After heterospecific transplantations of quail limb bud pieces, or other tissues containing blood vessels, into the limbs or the coelomic cavity, the immunohistochemically stained endothelial cells of the quail were found to invade the chick host vessels, favouring the arteries. Within these vessels the endothelial cells regularly reach the host aorta, where they contribute to the endothelium on the ipsilateral side. It is concluded that the endothelial cells activity migrate, because microinjections of a synthetic peptide which contains the RGD-sequence and mimics fibronectin, stop the invasion of endothelial cells.

Smooth muscle cells and fibroblasts of the coronary arteries derive from epithelial-mesenchymal transformation of the epicardium.

Previous research has revealed that cells contributing to coronary vascular formation are derived from the dorsal mesocardium, however, the fate of these cells during consecutive stages of heart development is still unclear. We have conducted a study regarding the recruitment of vascular components and the subsequent differentiation into mature vessel wall structures with the aid of immunohistochemical markers directed against endothelium, smooth muscle cells, and fibroblasts. The proepicardial organ including an adhering piece of primordial liver of quail embryos (ranging from HH15 to HH18) was transplanted into the pericardial cavity of chicken embryos (ranging from HH15 to HH18). The chicken-quail chimeras (n=16) were harvested from the early stage of endothelial tube formation (HH25) to the late stage of mature vessel wall composition (HH43). Before HH32 endothelial cells have invaded the myocardium to give rise to yet undifferentiated coronary vessels. These endothelial cells are not accompanied by other non-endothelial cells. The superficial epicardial layer changes from a squamous mesothelium into a cuboid epithelium preceding media and adventitia formation. Subsequently, a condensed area of mesenchymal cells delaminates from the cuboidal lining extending toward the vessel plexus. Around the coronary arteries, these mesenchymal cells differentiate into smooth muscle cells or fibroblasts as shown by immunohistochemical markers. We conclude that epithelial-mesenchymal transformation of the epicardial lining delivers the smooth muscle cells and fibroblasts of the coronary arterial vessel wall. Molecules involved in epithelial transformation processes elsewhere in the embryo are also expressed within the subepicardial layer, and are considered to participate in inducing this process.

The formation of mesoderm and mesectoderm in 5- to 41-somite rat embryos cultured in vitro, using WGA-Au as a marker.

The formation of mesectodermal cells by the neural crest in 5- to 41-somite stage embryos was investigated experimentally in rat embryos cultured in vitro, using lectin-coated colloidal gold as a probe. This method labelled all ectodermal cells, among them neural crest, surface ectodermal placodal and epiblastic (primitive streak) cells. The neural crest provides the mesodermal compartment of the entire head region with cells, including the primitive cranial ganglia and the branchial arches. In the head region migration of neural crest cells over a great distance (long-distance migration) was not observed. In the trunk region neural crest derived cells were mainly found to form the primitive spinal ganglia and the sympathetic trunk, once again without long-distance cell migration. Structures and tissues that supposedly were derived from the primitive streak were hardly labelled with colloidal gold. Surface ectodermal placodes were not only found at the expected sites (e.g. epibranchial placodes) but also in the ectoderm covering the transverse septum and lateral abdominal walls.

The development of the myocardium and endocardium in mouse embryos. Fusion of two heart tubes?

The formation of the single heart tube by hypothetical fusion of two separately developed heart tubes is re-investigated, because this intricate process is ambiguously and often incompletely described. To gain a better insight into this problem ten mouse embryos ranging from 7.5 to 8.5 days of development (presomite to 6 somites) were serially sectioned (1 micron) and reconstructed graphically. Twenty mouse embryos of comparative ages, were studied by scanning electron microscopy. Two large embryonic mesodermal compartments, derived from the primitive streak, extend rostrally on either side of the embryonic axis, and meet in front of the buccopharyngeal membrane. In each compartment a coelomic cavity develops, splitting the mesoderm into a splanchnic and somatic layer. The splanchnic mesoderm differentiates into a layer of cuboidal splanchnic mesothelial cells (promyocardium) and a subjacent plexus of elongated endothelial cells (proendocardium). Before the 1-somite stage the left and right splanchnic mesoderm are separated in front of the buccopharyngeal membrane by a thickening of the yolk sac endoderm. The splanchnic mesoderm then fuses, forming a single horseshoe-shaped heart primordium consisting of a promyocardial layer and a subjacent vascular plexus. Until the 2-somite stage both coelomic cavities remain separated by a bilayer of squamous somatic mesothelial cells ('mesocardium'). The plexus of endothelial cells that forms the proendocardium, also seems to be the source of the lining of the vitelline veins, the pharyngeal arch arteries and the dorsal aortae. The relatively close adherence of endoderm to the medial part of the horseshoe-shaped heart primordium, combined with a bilateral accumulation of cardiac jelly, is suggestive of a double heart tube. However, promyocardium and proendocardium are both translocated as one horseshoe-shaped layer, thus fusion of the left and right parts of the heart primordium does not occur.

The extracellular matrix during neural crest formation and migration in rat embryos.

Changes in the distribution of extracellular matrix components have been investigated immunohistochemically during neural crest development in the rat. Inside the ectodermal epithelium basal lamina components are formed resulting in a separation of neurectoderm and epidermal ectoderm. Within the presumptive neural crest area fibronectin, hyaluronan and chondroitin sulphate become apparent. Upon subsequent neural crest migration the basal lamina becomes disrupted. As the neural crest cells take part in mesectoderm formation, fragments of the basal lamina remain attached to their surface, as is demonstrated with antibodies against laminin and collagen type IV. The extracellular matrix is therefore active both in the separation of neuroectoderm from epidermal ectoderm and in mesectoderm formation.

The formation of mesoderm and mesectoderm in presomite rat embryos cultured in vitro, using WGA-Au as a marker.

Presomite rat embryos cultured in vitro were injected with the cell marker wheat germ agglutinin-gold in order to find out whether the ectoderm already formed mesodermal cells. These labelled so-called mesectodermal cells were found in all embryos studied, ranging in age from 8.7 to 9.3 days post coitum. In embryos younger than 9.0 days, the entire head fold ectoderm produced mesectodermal cells. From 9.0 days onwards, the neural crest and surface ectoderm placodes were recognizable as separate entities, both producing mesectodermal cells. The early onset of mesectodermal cell formation and the numerous and continuous manufacture led us to the conclusion that mesectodermal cells are deposited at their definitive location and that subsequent long-distance migration is unnecessary.

Maternoembryonic transfusion and congenital malformations: an experimental study using rat embryos.

In an experimental study, using an in vitro whole rat embryo culture, the effects of a maternoembryonic transfusion and immunologic interaction on the development of ten-day-old rat embryos (stages 8 to 10 somites) has been studied. Transplacental transfusion has been simulated by embryonic intracardiac microinjection of 0.1 to 0.5 microL immunologically active rat serum. After an incubation of 24 and 48 hours, respectively, the embryos were killed. All tested embryos have survived the incubation period. On microscopic examination of the tested embryos those that were taken from the incubator after 24 hours showed no signs of pathogenic cell degeneration, while the embryos that were taken from the incubator after 48 hours all had localized lesions with pathogenic cell degeneration in one or multiple major structures. The neurectoderm and endoderm seem to be the most sensitive tissues in this period of organogenesis. The results suggest that immunologic reaction to transplacental transfusion of maternal serum may lead to congenital malformations.