Relaxin as a protective substance in preservation solutions for organ transplantation, as shown in an isolated perfused rat liver model.

Reperfusion injury, a well-known problem in organ transplantation, results from multiple pathologic mechanisms, including platelet/mast cell activation and peroxidation of cell membrane lipids. Relaxin was originally described as an insulin-like hormone produced in the ovaries during pregnancy. It causes vessel dilation and inhibition of platelet and mast cell activation. The present study investigated the protective effect of relaxin against reperfusion injury in liver tissue. We used a model of isolated perfused rat liver to simulate liver transplantation. Organ preservation was performed identical to human transplantation in 20 male Wistar rats. During preservation we applied 64 ng/mL relaxin. In contrast controls (n = 10) had no relaxin treatment. To quantify cell damage, we measured malonyldialdehyde (MDA; end product of lipid peroxidation) and myeloperoxidase activity (MPO; marker for accumulation of neutrophil granulocytes) in the perfusates. The livers were examined immunohistochemically for the same parameters. Relaxin as an additional substance in preservation solutions decreased perfusate MPO and MDA levels by up to 30%, as shown by immunohistochemistry. Our preliminary data suggested that relaxin is a promising agent to reduce hepatocyte damage caused by ischemia-reperfusion injury. Quantitative analysis of MDA and MPO levels in the perfusate is the subject of an ongoing study.

Effect of bone sialoprotein and collagen coating on cell attachment to TICER and pure titanium implant surfaces.

To improve integration between implants and biological tissues, this study compared bone sialoprotein (BSP) as a surface-coating material against the major organic and inorganic components of bone, collagen type I and hydroxyapatite (TICER). The expression of osteocalcin, osteonectin and transforming growth factor ss was evaluated using immunohistochemical staining procedures. The distribution patterns of osteoblasts on the surface of pure titanium with a smooth machined surface and a rough surface (TICER) were determined by image processing using confocal laser scanning microscopy. The results compared to uncoated control materials showed that, at all times investigated, the number of cells on the surface of the TICER and pure titanium samples differed significantly (P<0.1), demonstrating the superiority of TICER over pure titanium in this respect. For pure titanium implants, collagen-precoated surfaces were not beneficial for the attachment of bone-derived cells with the exception of day 3 in vitro (P<0.01). BSP-precoated implant surfaces displayed non-significantly higher numbers of settled cells. BSP-precoated implant surfaces were beneficial for osteoinduction as revealed by osteocalcin and osteonectin expression. BSP precoating of the rough TICER implant surface enhanced the osteoinductive effect much more than did collagen precoating. These results contribute to the consideration of at least two distinct pathways of osseointegration.

Implant surface coatings with bone sialoprotein, collagen, and fibronectin and their effects on cells derived from human maxillar bone.

The interaction between implant material and surrounding tissues is believed to play a fundamental role in implant success. Although bone sialoprotein (BSP) has been found to be osteoinductive when coated onto femoral implants, collagen and fibronectin are the most used compounds for preparation of pre-coated cell culture slides at present. In this study, the support of BSP-, collagen- and fibronectin-coated and non-coated implant material for the development of adult human maxillar bone in vitro was studied and compared. The expression of bone turnover markers like BSP and osteocalcin as well as osteonectin, transforming growth factor beta (TGF-beta) and CD90 during different time periods of cell cultivation (3, 5, 10, 15, 20 and 25 days) was visualized immunohistochemically. The distribution patterns of the cells were examined on a rough surface of the titanium-hydroxyapatite dental implant material TICER and on a total smooth surface of the technical implant material glimmer. Significantly different values were found for glimmer at the 15. and the 20. Div, exclusively, indicating that a smooth surface was more improved than a rough ceramic surface by pre-coatings. The White-test using rankings of the median values gave evidence for BSP-coatings at position 1 followed by collagen. Our experiments were designed to use very low concentrated BSP coating solution with the aim to reduce the healing time with a minimal effort and minimal risks for the patients.

Wisteria floribunda agglutinin labeling patterns in the human cortex: a tool for revealing areal borders and subdivisions in parallel with immunocytochemistry.

Wisteria floribunda agglutinin (WFA) is a lectin that labels selectively N-acetylgalactosamines beta 1 (GalNAc beta 1-3 Gal) residues of glycoproteins within the extracellular matrix of the neurons, and has been identified as a specific marker for functionally different cortical areas of the rodent brain. Here we report that WFA-binding sites can be used for the characterization of cortical areas and their subdivisions of the immersion-fixed human brain. WFA-binding showed an area-specific distribution pattern within areas 1, and 3a-3b of the somatosensory cortex as well as in the primary motor areas 4a-4p. The WFA-binding labeled stripes of 150-175 microm width at intervals of 800-1000 microm within the motor cortex but not in the somatosensory cortex. At the cellular level, differences in staining intensities among certain cell types were evident among WFA-positive glial cells. WFA binding seems to be a useful marker to reveal areal borders and function related intraareal specializations in combination with immunocytochemical techniques.

Nitric oxide synthase isoenzymes during in vitro development of rat neuronal and human non-neuronal cells.

We studied the expression of neuronal (n), endothelial (e) and inducible (i) nitric oxide synthase (NOS) in cell cultures of rat mesencephalic neurons (embryonic day 14), human keratinocytes from juvenile epidermis, human endothelial cells from juvenile coronary arteries, and human osteoblasts. All cell types were cultured for 5, 10 or 15 days. During proliferation (round cells without processes), the intracellular distribution and the intracellular amount of the calcium-dependent NOS isoforms (n- and e-NOS) did not change whereas the calcium-independent i-NOS changed from a cytosolic distribution pattern to compartmentalized distribution. A striking decrease of i-NOS immunoreactivity was measured by means of image analysis. Our results support the opinion that i-NOS acts as a switch between proliferation and differentiation of cells.

Light and confocal laser-scanning microscopical evidences for complementary patterns of glial fibrillary acidic protein and Wisteria floribunda agglutinin labeled structures in human and rat brain.

We investigated the pattern of glial fibrillary acidic protein (GFAP) and Wisteria floribunda agglutinin (WFA) labeled structures in the superior colliculus and in the somatosensory cortex of humans and rats of different age groups using immunohistochemical methods, light and confocal laser-scanning microscopy. We never found a double labeling of WFA and GFAP positive structures neither in the superior colliculus nor in the cortex of both man and rat. The complementary pattern of WFA and GFAP labeling was present both at the macroscopic and microscopic level. We found a clear prevalence of either WFA or GFAP expression in the arborization of the astrocytes as well as in the pattern of lamination.

Retinoblastoma protein in microphthalmic mice.

A microphthalmic strain of mice was used to study immunoresponse of the retinoblastoma protein. Comparing wild-type, heterozygote and homozygote microphthalmic eyes, we found an increasing labelling of phosphorylated retinoblastoma protein (pRb) in the retinal pigment epithelium. Additionally, microphthalmic eyes expressed pRb in the neuroepithelium. Especially rosettes were strongly labelled.

Neuronal and inducible nitric oxide synthase distribution in retinas of microphthalmic mice.

A study about the distribution of neuronal nitric oxide synthase (n-NOS) and inducible NOS (i-NOS) was performed in microphthalmic retinas at postnatal day (pd) 2 and pd 7. Neither n-NOS nor i-NOS immunoresponse could be detected at pd 2. At pd 7, i-NOS activity was revealed in the retinas of mi/mi littermates but not in those of heterozygotes or wild-type littermates. The expression of n-NOS corresponded to the postnatal retinal development. Retinal rosettes were strongly labelled.

Dendritic organization of neurons of the superior colliculus in animals with different visual capability.

The aim of the study was to compare several morphological characteristics of neurons in the superficial layers of the superior colliculus in diurnal and nocturnal mammals with different visual specialization. Thus, we investigated the rat (Rattus norvegicus), a nocturnal animal; the tree shrew (Tupaia glis), a diurnal animal, and the Mongolian rodents, Microtus brandti (nocturnal) and Alticola barakshin (diurnal). The investigation was focused on the study of the organization and extent of dendrites of Golgi-impregnated projection neurons, which were divided in two classes: narrow-field and wide-field cells. We determined that the ratios between the volumes of dendritic fields of the investigated neuronal types and the total volume of the superior colliculus differed to a great extent between the different species. The tree shrew had the largest superior colliculus and the smallest wide-field neurons, while the rat had the largest wide-field neurons. As for the Mongolian rodents, we provided the first description of superior colliculus neurons. The day-active animal Alticola barakshin was found to have a 50% larger volume of the superior colliculus than that of the night-active animal Microtus brandti, and the size of the dendritic field of both wide-field neurons and narrow-field neurons was smaller than that of Microtus brandti. Electron microscopic investigation of wide-field neurons performed in the rat revealed only a few symmetric synaptic contacts on the arborizations of distal and terminal dendrites and numerous asymmetric synapses on the dendritic stem. Our findings support the hypothesis that whereas the narrow-field neurons are relay neurons in the retino-tecto-thalamic pathway of the visual system, the wide-field neurons may play additional roles in the retino-tecto-reticulo-spinal system.

Projection neurons in the superficial layers of the superior colliculus in the rat: a topographic and quantitative morphometric analysis.

The present study deals with qualitative and quantitative investigations in the superior colliculus of the rat. Tracer studies were correlated with Nissl staining to calculate the quantitative ratio between projection neurons and interneurons in the upper three layers of the superior colliculus. In order to reveal the projections from the superior colliculus, the first group of rats received injections of the tracer FluoroGold into the nucleus lateralis posterior thalami, the lateral geniculate body, the nucleus parabigeminalis, and the predorsal bundle. Commissural connections between the superior colliculus were traced in a second group of animals, which received Biocytin and FluoroGold injections in the upper layers of the right superior colliculus and small deposits of the carbocyanine tracer DiI in the deeper layers of the left superior colliculus. Additionally, double-labelling with FluoroGold tracing and the histochemical detection of NADPH-diaphorase activity was carried out to distinguish between projection neurons and interneurons. These experiments showed that 66% of the neurons within the superficial layers of the superior colliculus were represented by ascending projection neurons, whereas only 2-3% could be identified as descending neurons. Ascending neurons were scattered throughout the three laminae and descending neurons were localized in a cluster-like pattern. Approximately 2-3% of the neurons in the superficial layers were found to be commissural and interlayer neurons which were represented by an identical cell type, since both transcommissural and interlayer processes were originated from their somata. The somata of these commissural-interlayer neurons were all located in the mediorostral part of the superior colliculus and contained NADPH-diaphorase activity. The axon terminals of the interlayer-commissural neurons formed net-like structures which surrounded neuronal somata within the ipsilateral deep layers and within the contralateral upper layers of the superior colliculus, respectively.

Prostacyclin synthase is localized in rat, bovine and human neuronal brain cells.

Using a new polyclonal antibody against prostacyclin (PGI2)-synthase this enzyme was shown to be present in neuronal cells of bovine, rat and human brain, most abundantly in Purkinje cells of the cerebellum and cortical neurons, but not in glial cells. Western blots confirmed the specificity of the antibody and applied to enriched neuronal and astrocyte cultures supported these immunohistochemical data. It was further shown that staining with an anti-nitrotyrosine antibody was positive for PGI2-synthase containing cells. Possible physiological and/or pathophysiological functions of the enzyme in brain are discussed.

Neuronal and glial structures of the superficial layers of the human superior colliculus.

We studied neuronal and glial elements in the superficial layers of the human superior colliculus by means of Nissl stains, Golgi impregnations, histochemical demonstration of NADPH-d activity and immunohistochemistry for glial fibrillary acidic protein (GFAP) in astrocytes. The glia-neuron interface was visualized with Wisteria floribunda agglutinin (WFA), which is a marker for perineuronal nets. The laminar pattern and the morphology of the major cell types closely resembled that found in other species although the thickness of the stratum zonale varied and the diversity of interneurons was greater than in other mammals. Furthermore, the stratum griseum superficiale showed a characteristic clustering of cells, the surfaces of which were intensely labeled by WFA. The clusters disappeared when GFAP expression increased.

Region- and age-dependent variations of muscle fibre properties.

The cytophotometric-morphometrical analysis of extensor digitorum longus and soleus muscles of 2.5 and 18 months old rats revealed regional and age-dependent differences in fibre type distribution, fibre area and fibre type related-enzyme activities which characterize contractility and metabolic profile. Variations along the longitudinal axis from the origin to the insertion and along three transversal axes from superficial to deep were found dependent on the muscle investigated. For example, the fibres of extensor digitorum longus muscle showed increased contractile and glycolytic capacities near insertion and the fibres of soleus muscle increased oxidative capacity in its middle part. Furthermore, the contribution of the fibre type that is dominant in a muscle (fast-glycolytic fibre type in extensor digitorum longus and slow-oxidative fibre type in soleus muscle) to the total number of fibres increased from origin to insertion by 15 and 30%, respectively. Along the superficial-deep axes the oxidative capacity of all fibres increased, the most in fast fibres of the soleus muscle by approximately 50%. In soleus muscle, a decrease of cross areas of all fibre types from superficial to deep was found, correlating negatively with the succinate dehydrogenase activity of the fibres. In extensor digitorum longus muscle the change in cross areas of slow-oxidative and fast-oxidative glycolytic fibres was dependent on the position of the transversal axis in the muscle. The results suggest that distribution patterns of fibre types and the metabolic make up of individual muscle fibres are adapted on the basis of local functional demands. In both muscles, higher numbers and increased oxidative capacity of fast-glycolytic fibres were found during ageing, but variations from superficial to deeper regions were irrespective of age.

Cytoarchitecture of the tectum opticum in the Japanese quail.

The cytoarchitecture of the optic tectum of the Japanese quail, Coturnix coturnix japonica, was studied using the Golgi-Kopsch method, parvalbumin, calbindin and GABA immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry. Our results reveal a large number of different types of interneurons in the quail tectum opticum, only part of which are described in the chick or pigeon. Application of parvalbumin and calbindin immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry reveals the following lamination pattern: The stratum opticum, stratum griseum centrale and stratum album centrale remain unstained, while the laminae of the stratum griseum et fibrosum superficiale exhibit a roughly complementary staining pattern of calbindin (laminae c, d, e, f, g, i) and parvalbumin (laminae a, h, i). Nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry yields a dense band in lamina i. The Golgi material reveals the following cell types in the stratum griseum et fibrosum superficiale: marginal cells in the stratum opticum and in lamina h and i, horizontal cells in laminae a and c, large and small radial cells in laminae b, d, h and i, multiform cells in lamina b, bitufted cells in lamina d and e, large pear-shaped cells in lamina g, wide-field cells in lamina j, and stellate cells in lamina j and in the stratum griseum centrale. We consider horizontal cells, bitufted cells, multiform cells and small radial cells to be GABAergic interneurons of the stratum griseum et fibrosum superficiale which seem to be more numerous than in the pigeon tectum opticum. Golgi impregnation and injection of Phaseolus vulgaris leucoagglutinin into the pretectal nucleus lentiformis yielded regularly distributed clusters of telodendra of pretectal axons in lamina d of the stratum griseum et fibrosum superficiale, which are identical in shape and position with axon plexus revealed by Golgi staining.

Aberrant morphology of Müller glial cells in retinas of a microphthalmic mouse strain.

A microphthalmic mouse strain was used to study retinal glial cells during postnatal development of the retinal malformations. Glia was demonstrated with immunohistology using antibodies against vimentin or glial fibrillary acidic protein. To identify proliferating cells the bromodeoxyuridine technique was applied. Our results support the hypothesis that precursors of Müller cells may be involved in early stages of retinal malformations.

Conversion of biocytin labelled cells and structures for the confocal laser-scanning method.

The method for converting biocytin preparations of brain sections fills a gap in the application of confocal laser-scanning microscopy. Both neuronal and non-neuronal structures are converted. The background remains free of staining. The protocol can be applied to old and already existing biocytin-(diaminobenzidine)-nickel preparations which are then made accessible to evaluation with the laser-scanning microscope by the substitution of nickel with silver-gold. Sodium thiosulphate is used to remove the unbound silver. The reflection image of the laser-scanning microscopy provides more information than the transmission image.

24-hour rhythmicity of NADPH-diaphorase activity in the neuropil of rat visual cortex.

In the present study we examined the daytime-dependent alterations of nitric oxide synthase in the visual cortex of the rat. For this purpose, the activity of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), an enzyme equivalent to nitric oxide synthase, was measured histochemically in rat visual cortex at 0600, 1200, 1800, and 2400 h using a photometric scanning method. Our results show day-time-dependent changes of the NADPH-d activity in the neuropil of the visual cortex. This was highest at 0600 h and decreased between 1200 h and 1800 h (unimodal profile of circadian activity). The number of NADPH-d-positive neuronal somata was not found to vary at the different time points.

Corpus callosum axons in the developing visual cortex of the gerbil revealed by DiI and Fluorogold.

The distribution of commissural neurons and terminals was revealed by DiI and Fluorogold injections in gerbils. The distribution of corpus callosum projections in visual cortical areas changes significantly during postnatal development of the gerbil. On postnatal days 5, 8 and 10, the animals do not show callosal projections. Widespread callosal endings are developed by postnatal day 15. This diffuse projection concentrates in strips at the border of area 17/18A in adult animals (postnatal day 20 and older).

Hypoxia-dependent changes of enzyme activities in different fibre types of rat soleus and extensor digitorum longus muscles. A cytophotometrical study.

Using cytophotometry activity changes of succinate dehydrogenase, glycerol-3-phosphate dehydrogenase and myofibrillar adenosine triphosphatase were measured in 3 fibre types of soleus and extensor digitorum longus muscles under normal and experimental conditions. Fibres were typed by means of cytophotometrical data into slow-oxidative, fast-oxidative glycolytic and fast-glycolytic ones. After experimental hypoxia of 20 min duration a significant increase of enzyme activities was observed especially in slow-oxidative and fast-oxidative glycolytic fibres of both muscles, e.g. succinate dehydrogenase activity increased by 21% in these fibres of soleus muscle and by 23-26% in these fibres of extensor digitorum longus muscle. Moreover, an increase of glycerol-3-phosphate dehydrogenase activity by 10% in slow-oxidative fibres and by 28% in fast-oxidative glycolytic fibres and a 10-12% increased ATPase activity in all fibres of extensor digitorum longus muscle were measured. Treatment with Ginkgo biloba extract for 3 months before exposure to hypoxia resulted in increased adenosine triphosphatase activity in all fibres of both muscles and in decreased succinate dehydrogenase activity of slow-oxidative and fast-oxidative glycolytic fibres of extensor digitorum longus muscle. These results could be interpreted as a protective effect of Ginkgo biloba extract.

Autoradiographic and electron microscopic studies of retinal rosettes in genetically microphthalmic mice.

We report results obtained in the microphthalmic strain of mice 944. Heterozygotes appear normal, but they produce litters in which typically between 2 and 5 offspring exhibit microphthalmia and rosettes in the retina and the optic nerve. As a result these animals are blind. Our investigations using 3H-thymidine for autoradiographic estimation of postnatal cell proliferation and electron microscopy for detailed observation of the morphology of the rosettes show an increased proliferation of neuronal cells (more than 200%), especially in rosettes. In the rosettes a centrally located lumen exists in which cytoplasmatic processes can be found. In ultrastructural investigations these processes were identified as residues of the outer segments of photoreceptor cells.