Frank Nürnberger

Detection of Inducible Nitric Oxide Synthase and Vascular Endothelial Growth Factor in Choroidal Neovascular Membranes

Vascular endothelial growth factor (VEGF) is among the cytokines which have been implicated in the pathogenesis of choroidal neovascularization secondary to age-related macular degeneration (ARMD). There is, however, evidence that intercellular signaling molecules, such as nitric oxide (NO), are involved in this process. NO is synthesized via the inducible isoform of NO synthase (iNOS), which is expressed after induction by cytokines. In the current study, we investigated whether VEGF and iNOS are coexpressed in choroidal neovascular membranes (n = 7) from patients with ARMD. Immunohistochemistry was performed on cryosections with anti-iNOS and anti-VEGF. Moderate to intense immunostaining for iNOS and VEGF was observed in retinal pigment epithelial cells, macrophages, and in spatial relation to vessel walls. As scored by light microscopy, we found a significant correlation between immunoreactivity for VEGF and iNOS (p < 0.0341) in vascular endothelial cells. Our study supports a significant role for iNOS in the pathogenesis of neovascularization and membrane growth in ARMD. Moreover, our findings suggest a possible relationship between NO and VEGF in the regulation of pathologic angiogenesis in this disease.

The neuroendocrine system in hibernating mammals: present knowledge and open questions

The present review describes the distribution and the function-dependent reactivity pattern of those peptidergic and aminergic components of the neuroendocrine system of hibernating mammals that have been studied by histological, pharmacological and physiological techniques. Particular attention has been paid to the intrinsic connectivity of the peptidergic apparatus and its input systems. Since the reactivity patterns of the neuroendocrine system show remarkable fluctuations in relation to the various stages of hibernation and euthermia, these fluctuations have been analyzed with respect to (1) their causative role in the regulation of hibernation and (2) their secondary response to physiological changes during hibernation. The authors investigations described in this review have mainly been performed in European hedgehogs (Erinaceus europaeus), European and golden hamsters (Cricetus cricetus, Mesocricetus auratus), dormice (Glis glis), and in Richardsons and Columbian ground squirrels (Spermophilus richardsonii, Spermophilus columbianus), by the use of light- and electron-microscopic immunocytochemistry and histochemistry, in situ hybridization, radioimmunoassays and stereotaxically guided application techniques. These experiments were also performed in hypothermic animals. The (partially published) results obtained by the author and his associates are reviewed with reference to the body of evidence found in the recent literature. With respect to their reactivity patterns, several neuropeptide and transmitter systems can be regarded as candidates for control systems of hibernation. Neuronal complexes immunoreactive for endogenous opiates, in particular enkephalin, and also for vaspressin, somatostatin, substance P, corticotropin-releasing factor and serotonin are probably involved in the neuroendocrine control of hibernation.

The dispersed cell culture as model for functional studies of the subcommissural organ: preparation and characterization of the culture system

The subcommissural organ (SCO) is an enigmatic secretory gland of the brain, which is believed to be derived from ependymal (glial) precursor cells. We here developed a dispersed cell culture system of the bovine SCO as an approach to functional analyses of this brain gland. Tissue of the bovine SCO obtained from the slaughterhouse was papain dissociated either directly after dissection or after preparation of SCO explants. The latter had been maintained for 4-6 weeks in organ culture. The dispersed cells were cultured for up to 14 days and continuously tested for their secretory state by immunostaining of their secretory product. With respect to the morphology of the SCO cells (shape, processes, nucleus), no difference was found between the culture of freshly dissociated SCOs and that of dissociated SCO explants. In all cases, the dissociation caused a dedifferentiation; typical elongated cells were formed increasingly after 1 day of culture. Thereafter, only the cellular size increased, whereas the shape and the viability of the cells remained unchanged. Proliferating SCO cells were never observed. The culture obtained from fresh SCO tissue contained more glia cells and fibrocytes than the culture prepared from SCO explants. The proliferation of glia cells and fibrocytes was suppressed by blocking the mitotic activity with cytosine-beta-D-arabino furanoside (CAF). The cytophysiological features of the cultured dispersed cells of both origins did not differ as demonstrated by classical histology, by immunocytochemistry for the secretory products of the SCO, by the characteristics of calcium influx into the cytoplasm ([Ca2+]i) and cyclic adenosine monophosphate (cAMP) after stimulation with adenosine-5-triphosphate, substance P or serotonin, and by the activation of the transcription factor cAMP-responsive element-binding protein. Because of the maintenance of their viability, their capacity to release the secretory product into the culture medium, their receptive capacity, and their signal transduction pathways, we conclude that the dispersed cell culture system, especially that obtained from SCO explants, represents an appropriate and useful model for functional studies of the mammalian SCO.

Needlestick injuries among German medical students: time to take a different approach?

Context  Medical students are at risk of occupational exposure to blood‐borne viruses following needlestick injuries (NSIs) during medical school. The reporting of NSIs is an important step in the prevention of further injuries and in the initiation of early prophylaxis or treatment. The objective of this study was to describe the mechanisms whereby medical students experience occupational percutaneous blood exposure through NSIs and to discuss rational strategies for prevention.

Appraising the methodological quality of cadaveric studies: validation of the QUACS scale.

Although systematic reviews are conducted in the field of anatomical research, no instruments exist for the assessment of study quality. Thus, our objective was to develop a valid tool that reliably assesses the methodological quality of observational cadaveric studies. The QUACS scale (QUality Appraisal for Cadaveric Studies) was developed using an expert consensus process. It consists of a 13‐item checklist addressing the design, conduct and report of cadaveric dissection studies. To evaluate inter‐rater reliability, a blinded investigator obtained an initial pool of 120 observational cadaveric studies. Sixty‐eight of them were selected randomly according to sample size calculations. Three independent researchers rated each publication by means of the QUACS scale. The reliability of the total score was estimated using the intraclass correlation coefficient (ICC). To assess agreement among individual items, margin‐free kappa values were calculated. For construct validity, two experts (an anatomist and an experienced physician) categorized the quality of 15 randomly selected studies as ‘excellent’ (4 points), ‘moderate to good’ (3 points), poor to moderate’ (2 points) or ‘poor’ (1 point). Kendalls tau rank correlation was used to compare the expert ratings with the scores on the QUACS scale. An evaluation of feasibility was carried out during the reliability analysis. All three raters recorded the duration of quality appraisal for each article. Means were used to describe average time exposure. The ICC for the total score was 0.87 (95% confidence interval: 0.82–0.92; P < 0.0001). For individual items, margin‐free kappa values ranged between 0.56 and 0.96 with an agreement of 69–97% among the three raters. Kendalls tau B coefficient of the association between expert ratings and the results obtained with the QUACS scale was 0.69 (P < 0.01). Required rating time per article was 5.4 ± 1.6 min. The QUACS scale is highly reliable and exhibits strong construct validity. Thus, it can confidently be applied in assessing the methodological quality of observational dissection studies.

Effects of neuroactive substances on the activity of subcommissural organ cells in dispersed cell and explant cultures

Abstract. The subcommissural organ (SCO), an ependymal (glial) circumventricular organ, releases glycoproteins into the cerebrospinal fluid; however, the regulation of its secretory activity is largely unknown. To identify neuroactive substances that may regulate SCO activity, we investigated immunocytochemically identified bovine SCO cells by means of calcium imaging. This analysis was focused on: (1) serotonin (5HT) and substance P (SP), immunocytochemically shown to be present in axons innervating the bovine SCO; and (2) ATP, known to activate glial cells. 5HT had no effect on the intracellular calcium concentration ([Ca2+]i), and its precise role remains to be clarified. SP elicited rises in [Ca2+]i in approx. 30% and ATP in even 85% of the analyzed SCO cells. These effects were dose-dependent, involved NK3 and P2Y2 receptors linked to G protein and phospholipase C (PLC) activation, and could not be mimicked by forskolin or 8-bromo-cAMP. In 50% of the SP-sensitive cells, the increases in [Ca2+]i comprised calcium release from thapsigargin-sensitive intracellular stores and an influx of extracellular calcium via protein kinase C (PKC)-induced opening of L-type voltage-gated calcium channels (VGCCs). In the remaining SP-sensitive cells, the increase in [Ca2+]i was caused exclusively by influx of extracellular calcium via VGCCs of the L-type. In all ATP-sensitive cells the increase in [Ca2+]i involved calcium release from thapsigargin-sensitive intracellular stores and a PKC-mediated influx of extracellular calcium via L-type VGCCs. Our data suggest that SP and ATP are involved in regulation of the activity of SCO cells.

Presence and functional significance of neuropeptide and neurotransmitter receptors in subcommissural organ cells.

The subcommissural organ (SCO) of mammals is innervated by several neuropeptide and neurotransmitter systems. So far, substance P (SP), oxytocin (OXT), vasopressin (VP), somatostatin (SOM), thyrotropin‐releasing factor (TRF), and angiotensin II (ANGII) were identified in neuropeptidergic input systems, and serotonin (5HT), gamma‐amino butyric acid (GABA), noradrenaline (NA), dopamine (DA), and acetylcholine (Ach) were neurotransmitters observed in systems afferent to the SCO. In the present report, based on literature data and our own investigations, we describe the occurrence of peptide and transmitter receptors in the SCO by means of autoradiographic and biochemical studies. Further, we summarize aspects of the signal transduction cascades possibly linked to different receptor types of the SCO; these studies included the use of calcium imaging (FURA‐2 technique), ELISA technique, and immunocytochemistry. Receptors were identified for adenosine, angiotensin II, imidazoline, glucocorticoids, mineralocorticoids, NA, and embryonic brain kinase. The studies on intracellular signal‐transduction indicated receptors for tachykinins and for ATP. In SCO cells, Ca++ and c‐AMP were identified to act as second messengers. As important transcription factor, cAMP‐/Ca++‐response element binding protein (CREB) was observed. Ach and NA did not show a significant effect on the subcommissural signal transduction. Microsc. Res. Tech. 52:534–540, 2001.

The somatostatin system of the brain and hibernation in the European hamster (Cricetus cricetus).

The depression of physiological processes characteristic of mammalian hibernation is precisely regulated by the central nervous system, especially by the neuropeptidergic apparatus of the hypothalamus. Because of inhibitory influences on neuronal circuits within the brain and suppressive effects on the metabolism via the endocrine axis, somatostatin has been implicated in the regulation of hibernation. The somatostatin system of the brain was investigated with immunocytochemistry, in situ hybridization, and radioimmunoassays in euthermic summer, euthermic winter, and hibernating European hamsters (Cricetus cricetus). Numerous somatostatin-immunoreactive perikarya were observed in the periventricular hypothalamic nucleus. The striatum, amygdala, and cortex contained only scattered immunoreactive perikarya. These entities also contained immunoreactive fiber profiles, although the highest density of immunoreactive fibers was found in the median eminence. Immunocytochemistry and radioimmunoassays showed that the number of somatostatin-immunoreactive perikarya and fibers and the content of somatostatin in the hypothalamus and the median eminence was conspicuously lower in euthermic winter animals than in euthermic summer animals. This decrease was more pronounced in hibernating specimens. In situ hybridization also demonstrated a decrease in the expression and synthesis rate of somatostatin in euthermic winter animals; again, this was even more dramatic in hibernating hamsters. These changes were less pronounced or non-significant in the extrahypothalamic somatostatin-immunoreactive perikarya and fiber systems, as shown by immunocytochemistry and radioimmunoassay, respectively.

Beta-adrenergic signal transduction in the hypothalamus of the European hamster: Relation with the seasonal hibernation cycle and the diurnal activity cycle

Mammalian hibernation, an adaptation to survive harsh winter conditions, is one of the most prominent seasonal rhythmic processes exactly regulated on a low metabolic level. Diurnal variations in vegetative physiology are missing during hibernation; however, a precisely working diurnal system is mandatory for both the proper initiation and termination of the annual hibernation phase and the periodical arousal reactions. Biorhythms and the vegetative physiological processes connected with hibernation are, among others, controlled by hypothalamic noradrenaline systems. In this study, the density, binding capacity, and relative proportions of β1‐ and β2‐adrenergic receptors (AR) within the hypothalamus of: 1) motorically inactive summer; 2) motorically active summer; 3) aroused, motorically active winter; and 4) deeply hibernating winter European hamsters (Cricetus cricetus) were studied. For further analysis of the β‐adrenergic signal transduction cascade, the activity of adenylyl cyclase (AC) was measured by formation of cAMP in controls, after stimulation of G proteins, or after forskolin stimulation without or in presence of manganese ions. While β1‐ and β2‐AR subtypes were nearly equally abundant (50% β1:50% β2) in active summer, inactive summer, and hibernating hamsters, a significant redistribution in favor of β2‐AR occurred after arousal (40% β1:60% β2). The activity of AC was much higher in active summer hamsters than in inactive summer, aroused winter, and hibernating winter hamsters. When AC was stimulated by guanylylimidophosphate [Gpp(NH)p], MnCl2, forskolin, or by forskolin in presence of MnCl2 instead of MgCl2, the potency to stimulate AC was found to show the following rank order: basal < Gpp(NH)p < MnCl2 ≤ forskolin + MnCl2 < forskolin.

Neuropeptides and Neurotransmitters in the Suprachiasmatic Nucleus: Relationship with the Hibernation Process

To elucidate the functional role of the suprachiasmatic nuclei (SCN) in control of hibernation, especially in timing of hibernation and arousal, the reactivity patterns of suprachiasmatic neuropeptide and neurotransmitter systems were studied in several species of mammalian hibernators by the use of histochemical and biochemical methods during different stages of hibernation and euthermia. Although the distribution patterns of the suprachiasmatic messenger systems of hibernating species did not differ from that in mammals not able to hibernate, their reactivity patterns were dramatically changing in relation with the stages of hibernation, arousal or euthermia. The messenger systems directly or indirectly transmitting light signals to the SCN (glutamate [GLU], pituitary-adenylyl cyclase activating peptide [PACAP], neuropeptide Y [NPY]) were almost inactive, whereas the systems originating from other entities within the brain (serotonin [5HT], substance P [SP], somatostatin [SOM], enkephalin [ENK]) remain active throughout the entire hibernation phase. Among the messenger systems intrinsic to the SCN, the reactivity of the gamma-amino butyric acid (GABA) system remains almost stable between hibernation and euthermia, whereas the reactivity of optic input relays (vasoactive intestinal peptide [VIP], gastrin-releasing peptide [GRP]) was drastically decreased and that of output relays (arginine vasopressin [AVP]) was increased during hibernation. These data together with results on regulatory processes in hypothalamic and basal forebrain structures emphasize a prominent role of the SCN in timing of initiation and termination of hibernation.