Hyaluronic acid coating of gold nanoparticles for intraocular drug delivery: Evaluation of the surface properties and effect on their distribution.

Due to the unique anatomical structure of the eye, ocular drug delivery is a promising delivery route for the treatment of several ocular diseases, such as the ocular neovascularization that contributes to diabetic retinopathy. This disease is triggered by inflammation, retinal ischemia, and/or deposits of advanced-glycation end-products (AGEs), as well as increased levels of vascular endothelial growth factor (VEGF), interleukins, or reactive oxygen species (ROS). Gold has unique antioxidant and antiangiogenic properties and can inhibit angiogenic molecules. Furthermore, gold nanoparticles (GNPs) are not only biocompatible, they are easy to synthesize, they absorb and scatter visible light, and they can be made with precise control over size and shape. GNPs are an excellent candidate for ocular drug delivery because they can be conjugated to an extraordinarily diverse array of different biomolecules, and surface functionalization can improve the mobility of GNPs across the physiological barriers of the eye, such as the vitreous humour or the inner limiting membrane. For this purpose, we employed low molecular weight hyaluronan (HA) to increase the mobility of the nanoparticles as well as target them to HA receptors that are expressed in different cells of the eye. In this study, the combination of gold and HA enhanced the stability of the whole carrier and promoted their distribution across ocular tissues and barriers to reach the retina. Moreover, analysis in vitro, ex vivo, and in ovo revealed the protective and antiangiogenic effect of GNPs as inhibitors of AGEs-mediated- retinal pigment epithelial cell death and neovascularization. We demonstrated that conjugation with HA enhances GNP stability and distribution due to a specific CD44 receptor interaction. The capacity of HA-GNPs to distribute through the vitreous humour and their avidity for the deeper retinal layers ex vivo, suggest that HA-GNPs are a promising delivery system for the treatment of ocular neovascularization and related disorders.

The anemia of the newborn induces erythropoietin expression in the developing mouse retina.

The hematopoietic hormone erythropoietin (Epo), regularly produced by the kidneys and the liver, is also expressed in neuronal tissue, where it has been found to mediate paracrine neuroprotective effects. In most studies exploring the rescue effects of Epo, apoptosis was exogenously induced by different cell death stimuli. Herein, we set out to study the expression and function of Epo in physiologically occurring apoptosis in a model of retinal development. We made use of an organotypic retinal wholemount culture system that resembles the physiological in vivo situation with cell connections still retained. Epo mRNA expression in the retina, liver, and kidney showed a significant increase during early development, coinciding with the anemia of the newborn. In the retina of Epo-green fluorescent protein transgenic mice, Epo-expressing cells were identified and found to be distributed in the retinal ganglion cell layer. Treatment of retinal wholemount cultures with recombinant Epo resulted in a significant decrease of apoptotic ganglion cells as well as photoreceptor cells throughout retinal development. Moreover, transforming growth factor-beta-induced apoptosis was completely antagonized by Epo when both factors were simultaneously applied. Investigations on the signaling pathway revealed a decrease in Bax mRNA levels in Epo-treated retinal cells. We conclude that Epo exerts wide and prolonged neuroprotective activity in physiologically occurring apoptosis and thus contributes to proper retinal development.

Immunohistochemical evaluation of matrix molecules associated with wound healing following treatment with an enamel matrix protein derivative in humans.

Application of enamel matrix protein derivative (EMD) onto a debrided and conditioned root surface has been shown to promote periodontal regeneration in animals and humans. However, until now there is virtually no information from humans describing the expression of different matrix molecules in the newly formed periodontal tissues following treatment with EMD. This study investigated immunohistochemically in humans the expression of matrix molecules associated with periodontal tissues reformed after treatment with EMD. Eight patients with intrabony defects were treated with EMD. Six months after surgery teeth together with some of their surrounding soft and hard tissues were removed, fixed in buffered formalin, decalcified in EDTA, and embedded in paraffin. Serial sections of 6 micro m were cut in mesiodistal direction. Sections were evaluated immunohistochemically by means of polyclonal antibodies against osteopontin, collagen I and collagen III. The original (non-treated) parts of the periodontium served as controls. In all specimens the healing resulted to a varying extent in formation of cementum, periodontal ligament and alveolar bone. In all specimens the expression of the investigated matrix molecules was stronger at the reformed than at the original sites. Osteopontin expression was most intense at the border near the newly formed cementum and bone. In the regenerated periodontal ligament, collagen I and III were localized throughout the entire periodontal ligament connective tissue. Within the newly formed PDL connective tissue the immunohistochemical staining appeared stronger for collagen III than for collagen I. The present findings suggest that (a) treatment of human intrabony defects with EMD creates an environment favourable for periodontal regeneration and, (b) in humans the healing and/or remodelling process of the reformed tissues may be followed immunohistochemically for a period of 6 months.

Plasma serotonin and histamine levels in hemodialysis-related pruritus are not significantly influenced by 5-HT3 receptor blocker and antihistaminic therapy.

BACKGROUND: Elevated plasma histamine levels are considered to play a part in the pathophysiology of hemodialysis-related pruritus. However, antihistaminic therapy often fails to provide sufficient relief. Elevated serotonin levels in patients on dialysis therapy have also been described but the effects of 5-HT3 receptor antagonists on hemodialysis-related pruritus remain controversial. METHODS: we conducted a study to determine plasma histamine and serotonin levels before and after treatment with 5-HT3 receptor antagonists (tropisetron 5 mg and ondansetron 8 mg) and an antihistamine (cetirizine 10 mg). Eleven hemodialysis patients with a history of pruritus participated in this study,10 healthy volunteers served as control group. RESULTS: Histamine and serotonin values were normal in patients and controls. Treatment with cetirizine did not significantly reduce histamine levels in patients or in controls. Tropisetron and ondansetron likewise did not alter serotonin levels in patients. Tropisetron treatment did not significantly change serotonin levels in controls. CONCLUSION: Histamine and serotonin are no major mediators of pruritus in hemodialysis patients. Elevated histamine levels are occassionally seen and may be due to the increased mast cell number found in a subgroup of hemodialysis patients. Our findings explain the only marginal relief of antihistamines and the controversial antipruritic effect of serotonin receptor antagonists in hemodialysis-related pruritus.

Immunohistochemical evaluation of matrix molecules associated with wound healing following regenerative periodontal treatment in monkeys.

The aim of the study was to investigate immunohistochemically the expression of matrix molecules associated with periodontal tissues reformed after regenerative periodontal treatment. Chronic intrabony defects were treated with guided tissue regeneration, enamel matrix proteins, the combination of both, or access flap surgery. Five months after healing, the animals were killed, and the healed periodontal tissues were evaluated immunohistochemically by means of polyclonal antibodies against osteopontin, collagen I, and collagen III. The intact (nontreated) parts of the periodontium served as controls. As a general observation, the staining for all investigated matrix molecules appeared to be stronger within the regenerated tissues than in the intact ones. The results failed to reveal any differences in terms of staining intensity or distribution pattern of investigated matrix molecules between the four different treatments. Osteopontin expression was most intense at the border near the newly formed cementum and bone. In the regenerated periodontium, collagens I and III were localized throughout the entire periodontal ligament connective tissue. In the regenerated periodontal ligament, collagen III displayed more intense staining than collagen I. The present results suggest that: (1) even after a 5-month period following surgical periodontal therapy, extracellular matrix molecules associated with wound healing and/or remodelling are more strongly expressed in regenerated than in intact tissues and (2) once an environment for periodontal regeneration has been created, the expression of extracellular matrix molecules associated with the healing process seems to display the same pattern, irrespective of treatment modality.

TGF-beta modulates programmed cell death in the retina of the developing chick embryo.

Programmed cell death (PCD) is a key phenomenon in the regulation of cell number in multicellular organisms. We have shown that reduction of endogenous transforming growth factor beta (TGF-beta) prevents apoptotic PCD of neurons in the developing peripheral and central nervous system, suggesting that TGF-beta is an important mediator of ontogenetic neuron death. Previous studies suggested that there are other pro-apoptotic molecules, nerve growth factor (NGF) and brain-derived neurotrophic factor, that induce cell death in the nervous system. In the developing chick retina, NGF induces PCD by activation of the p75 receptor. We have studied the role of TGF-beta and its putative interdependence with NGF-mediated PCD in the chick retina. We found that TGF-beta is present in the developing chick retina during the period of PCD and is essentially required to regulate PCD of retinal cells. TGF-beta 2, TGF-beta 3 and the ligand-binding TGF-beta receptor can be detected immunocytochemically in the central retina, a region where apoptosis is most prominent during the early period of PCD. Application of a TGF-beta-neutralizing antibody to chick embryos in ovo resulted in a decrease in the number of TUNEL-positive cells and a reduction of free nucleosome levels. In terms of magnitude, reduction of PCD caused by the neutralization of endogenous TGF-beta was equivalent to that seen after anti-NGF application. Neutralization of both factors did not result in a further decrease in apoptosis, indicating that NGF and TGF-beta may act on the same cell population. Furthermore, neutralization of TGF-beta did not affect the expression of NGF or the p75-receptor. Our results suggest that TGF-beta and NGF are both required to regulate cell death in the chick retina in vivo.

Targeted mutations of transforming growth factor-beta genes reveal important roles in mouse development and adult homeostasis.

Transforming growth factors-beta (TGF-beta) are multifunctional molecules with profound biological effects in many developmental processes including regulation of cell proliferation, differentiation, cell adhesion, skeletal development, haematopoiesis, inflammatory responses, and wound healing. To learn about the role of TGF-beta in vivo, phenotypes of targeted mutations of molecules within the TGF-beta signalling pathway, TGF-beta1, -beta2, -beta3, TGF-beta receptor (TbetaR-II) and the signalling molecules SMAD2, SMAD3 and SMAD4, are discussed in this review. The three individual TGF-beta mutants show distinct and only partially overlapping phenotypes. In mice, targeted disruption of the TGF-beta1 gene results in diffuse and lethal inflammation about 3 weeks after birth, suggesting a prominent role of TGF-beta in the regulation of immune cell proliferation and extravasation into tissues. However, just half of the TGF-beta1 (-/-) conceptuses actually reach partuition due to defective haematopoiesis and endothelial differentiation. Targeted disruption of both TGF-beta2 and TGF-beta3 genes results in perinatal lethality. TGF-beta2 null mice exhibit a broad range of developmental defects, including cardiac, lung, craniofacial, limb, eye, ear and urogenital defects, whereas TGF-beta3 gene ablation results exclusively in defective palatogenesis and delayed pulmonary development. The TbetaR-II null phenotype closely resembles that of TGF-beta1 (-/-) conceptuses, which die in utero by E10.5. Loss of SMAD2 or SMAD4 results in related phenotypes: the mutants fail to form an organized egg cylinder, lack mesoderm required for gastrulation and die prior to E8.5. Together, gene ablation within the TGF-beta signalling pathway supports the notion of a prominent role of TGF-beta during development.

Reduction of endogenous transforming growth factors beta prevents ontogenetic neuron death.

We show that following immunoneutralization of endogenous transforming growth factors beta (TGF-beta) in the chick embryo, ontogenetic neuron death of ciliary, dorsal root and spinal motor neurons was largely prevented, and neuron losses following limb bud ablation were greatly reduced. Likewise, preventing TGF-beta signaling by treatment with a TbetaR-II fusion protein during the period of ontogenetic cell death in the ciliary ganglion rescued all neurons that normally die. TUNEL staining revealed decreased numbers of apoptotic cells following antibody treatment. Exogenous TGF-beta rescued the TGF-beta-deprived phenotype. We conclude that TGF-beta is critical in regulating ontogenetic neuron death as well as cell death following neuronal target deprivation.

Embryonic and larval development in the caecilian Ichthyophis kohtaoensis (Amphibia, gymnophiona): a staging table.

Little is known about the developmental biology of caecilians-tropical, elongate, limbless, mostly fossorial amphibians that are members of the Order Gymnophiona. Ichthyophis kohtaoensis (Family Ichthyophiidae; southeast Asia) is an oviparous species in which maternal care of the clutch is provided. The clutch is laid in a burrow on land, and the embryos develop in their egg membranes, curved around a large yolk mass. Larvae are aquatic and exhibit characteristic features that are not present in the terrestrial adults. Because accurate descriptions of ontogenies and the establishment of standardized stages of embryonic and larval development are useful for both experimental and comparative embryology, a staging table for I.kohtaoensis was developed based on external morphological features. Development from the end of neurulation to metamorphosis was divided into 20 stages. Principal diagnostic features include development of the lateral line organs, formation of three pairs of external gills, development of the eyes, changes in yolk structure, changes in the structure of the cloacal aperture and growth of the tail, including the formation and regression of the tail fin. This study provides a comparison with descriptions of embryonic stages of I.glutinosus and Hypogeophis rostratus and with a recent staging table for the aquatic, viviparous caecilian Typhlonectes compressicauda, the only other caecilians for which reasonably complete ontogenetic information exists in the literature. Comparisons with established staging tables for selected frogs and salamanders are also presented.

Serotonergic neurons and processes in the adult and developing retina of Ichthyophis kohtaoensis (Amphibia; Gymnophiona).

Ichthyophis kohtaoensis is a tropical, limbless amphibian species with extremely small eyes (540 microm in adults). Adapted to a subterranean, burrowing mode of life, orientation and prey capture are predominantly guided by olfaction. The only visually guided behavior seems to be negative phototaxis. As electrophysiological single cell recordings have so far failed, immunohistochemical transmitter studies are a starting-point for a functional investigation of the visual system of this group of amphibians. In the present study, the organization and development of the serotonergic system have been examined in the retinae of embryonic, larval and adult I. kohtaoensis, using an antiserum against serotonin. Labeled somata are situated in the inner nuclear layer, presumably representing amacrine cells, and in the ganglion cell layer. However, some immunoreactive cells are located in the middle of the inner nuclear layer that send processes to both plexiform layers, probably representing bipolar cells. An additional type of immunoreactive soma, situated in the inner plexiform layer, have been found only in retinae of embryonic stages. Varicose serotonergic fibers form a diffuse plexus that covers the whole inner plexiform layer. Immunolabeled fibers can occasionally be demonstrated in the optic nerve head. During retinal development, the distribution and number of transmitter-expressing cells changes but no general reduction of the visual system is detectable. Adult stages still have a serotonergic system comparable to amphibians with a well-developed visually guided behavior indicating that the eyes of I. kohtaoensis although being of minor importance in a subterranean habitat, retain all the elements of functioning sense organs.

A double-label analysis demonstrating the partial coexistence of tyrosine hydroxylase and GABA in retinal neurons of ichthyophis kohtaoensis (Amphibia; gymnophiona)

Ichthyophis kohtaoensis is a limbless amphibian species with a subterranean mode of life and a predominantly olfactorily guided orientation. The only visually guided behavior seems to be negative phototaxis. As these animals possess extremely small eyes (only 540 microm in diameter in adults), functional investigations of the retina by electrophysiological single cell recordings have so far failed. Therefore, immunohistochemical transmitter studies constitute a starting point for a functional investigation of the caecilian retina. Previous immunohistochemical examinations have revealed immunoreactivity for gamma-aminobutyric acid (GABA), serotonin, dopamine and tyrosine hydroxylase (TH). The double-labeling experiments of the present study show that some TH-immunoreactive cells also express GABA-like immunoreactivity and some GABAergic neurons also label for TH immunocytochemistry, revealing a partial coexistence of TH and GABA in caecilian retinal neurons. On the one hand, these results contrast with previous reports, stating that in amphibians GABA-immunoreactive cells constitute a separate population from TH-positive neurons and colocalization is restricted to higher vertebrates. On the other hand, the findings indicate that a functional system which is under no strong selective pressure obviously has a long evolutionary persistence irrespective of its need for use.

Colocalization of serotonin and GABA in retinal neurons of Ichthyophis kohtaoensis (amphibia; Gymnophiona).

Ichthyophis kohtaoensis, a member of the limbless Gymnophiona, has a specialized subterranean burrowing mode of life and a predominantly olfactory-guided orientation. The only visually guided behavior seems to be negative phototaxis. As these animals possess extremely small eyes (only 540 microm in diameter in adults), functional investigations of single retinal cells by electrophysiological methods have so far failed. Therefore, the content and distribution of retinal transmitters have been investigated as indications of a functioning sense organ in an animal that is supposed to be blind. Previous immunohistochemical investigation of the retinal transmitter system revealed immunoreactivity for gamma-aminobutyric acid (GABA), serotonin, dopamine and tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthetic pathway. The present studies have been performed in order to determine a possible colocalization of serotonin and GABA in retinal neurons of the caecilian retina. Therefore retinal cryostat sections of various developmental stages have been investigated by the indirect fluorescence method. In single-label preparations, serotonin is localized to cells in the inner nuclear layer and the ganglion cell layer. GABA immunocytochemistry labels a variety of cell types in the inner nuclear layer as well as cell bodies in the ganglion cell layer. In double-label preparations, some of the serotonergic cells are found to express GABA immunoreactivity and some GABAergic neurons also label for serotonin immunocytochemistry. Thus, despite the fact that caecilians mainly rely on olfaction and are believed to have a reduced visual system, their retina exhibits a surprisingly "normal" distribution of neurotransmitters and neuromodulators, also typical of other anamniotes with a well-developed visual system, including the partial colocalization of serotonin and GABA at all developmental stages of I. kohtaoensis. These results indicate that a functional system that is under no strong selective pressure obviously has a long evolutionary persistence irrespective of its need for use.

Development and organization of the retinal dopaminergic system of Ichthyophis kohtaoensis (Amphibia; Gymnophiona).

Ichthyophis kohtaoensis, a member of the limbless Gymnophiona, has a specialized subterranean burrowing mode of life and a predominantly olfactory-guided orientation. The only visually guided behavior seems to be negative phototaxis. As these animals possess extremely small eyes (only 540 microm in diameter in adults), functional investigations of single retinal cells by electrophysiological methods have so far failed. Therefore, the content and distribution of retinal transmitters have been investigated as indications for a functioning sense organ in an animal that is supposed to be blind. In this study, the organization and development of the dopaminergic system have been examined in the retinae of embryonic, larval, and adult I. kohtaoensis, by using an antiserum against tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthetic pathway, and an antiserum against dopamine itself. Labeled somata are situated in the inner nuclear layer and in the ganglion cell layer. Dopamine-positive fibers form a dense diffuse plexus, that covers the whole inner plexiform layer, whereas tyrosine hydroxylase-immunoreactive processes show a tendency to arborize in a stratified manner. Tyrosine-hydroxylase-immunolabeled fibers can occasionally be observed in the optic nerve head of larval stages. During ontogenesis and larval development, the distribution of transmitter-expressing cells changes and their number decreases, but no general degeneration of the visual system is detectable. Adult Ichthyophis still have retinal transmitters, indicating that the eyes, although obviously playing a minor role in a subterranean ecological niche, retain all the elements of functioning sense organs.

[Sedimentation analyser--calibration and testing of a newly developed device for recording separation behavior of blood and other dispersed systems]. / Sedimentationsanalysator--Eichung und Testung eines neuentwickelten Gerätes zur Aufzeichnung des Entmischungsverhaltens von Blut und anderen dispersen Systemen.

A computer-aided sedimentation analyser is described, which can be used for the continuous monitoring of the separation of blood and other dispersed systems by recording the separation-dependent infrared transmission in the range between 100 xg to 700 xg. Up to eight samples can be measured simultaneously within a short period of time, and only a small amount of suspension is required (15 minutes, 350 microliters). For centrifugal acceleration more than 100 xg and a haematocrit range of between 0.2 and 0.7, the evolution of the height of the plasma column (HPC) over time is expressed in non-linear regression form by HPC(t) = HPC infinity*(1-e-kt) + c. The separation constant k is influenced by plasma viscosity, haematocrit, aggregability and erythrocyte deformability, is directly proportional to centrifugal acceleration, and declines in hyperbolic fashion with increasing haematocrit between 300 xg and 650 xg. The separation constant is closely related to the maximum velocity which, in fact, represents the sensitive parameter of separation. Thus, the sedimentation analyser can be applied as an alternative to the traditional measurement of erythrocyte sedimentation rate according to Westergren.