Ex Vivo Construction of an Artificial Ocular Surface by Combination of Corneal Limbal Epithelial Cells and a Compressed Collagen Scaffold Containing Keratocytes

We have investigated the use of a laminin coated compressed collagen gel containing corneal fibroblasts (keratocytes) as a novel scaffold to support the growth of corneal limbal epithelial stem cells. The growth of limbal epithelial cells was compared between compressed collagen gel and a clinically proven conventional substrate, denuded amniotic membrane. Following compression of the collagen gel, encapsulated keratocytes remained viable and scanning electron microscopy showed that fibres within the compressed gel were dense, homogeneous and similar in structure to those within denuded amniotic membrane. Limbal epithelial cells were successfully expanded upon the compressed collagen resulting in stratified layers of cells containing desmosome and hemidesmosome structures. The resulting corneal constructs of both the groups shared a high degree of transparency, cell morphology and cell stratification. Similar protein expression profiles for cytokeratin 3 and cytokeratin 14 and no significant difference in cytokeratin 12 mRNA expression levels by real time PCR were also observed. This study provides the first line of evidence that a laminin coated compressed collagen gel containing keratocytes can adequately support limbal epithelial cell expansion, stratification and differentiation to a degree that is comparable to the leading conventional scaffold, denuded amniotic membrane.

Diabetes mellitus-related morphoquantitative changes in the celiac ganglion neurons of the dog

Diabetes mellitus is the most common endocrine disturbance of domestic carnivores and can cause autonomic neurological disorders, although these are still poorly understood in veterinary medicine. There is little information available on the quantitative adaptation mechanisms of the sympathetic ganglia during diabetes mellitus in domestic mammals. By combining morphometric methods and NADPH-diaphorase staining (as a possible marker for nitric oxide producing neurons), type I diabetes mellitus-related morphoquantitative changes were investigated in the celiac ganglion neurons in dogs. Twelve left celiac ganglia from adult female German shepherd dogs were examined: six ganglia were from non-diabetic and six from diabetic subjects. Consistent hypertrophy of the ganglia was noted in diabetic animals with increase of 55% in length, 53% in width, and 61.5% in thickness. The ordinary microstructure of the ganglia was modified leading to an uneven distribution of the ganglionic units and a more evident distribution of axon fascicles. In contrast to non-diabetic dogs, there was a lack of NADPH-diaphorase perikarial labelling in the celiac ganglion neurons of diabetic animals. The morphometric study showed that both the neuronal and nuclear sizes were significantly larger in diabetic dogs (1.3 and 1.39 times, respectively). The profile density and area fraction of NADPH-diaphorase-reactive celiac ganglion neurons were significantly larger (1.35 and 1.48 times, respectively) in non-diabetic dogs compared to NADPH-diaphorase-non-reactive celiac ganglion neurons in diabetic dogs. Although this study suggests that diabetic neuropathy is associated with neuronal hypertrophy, controversy remains over the possibility of ongoing neuronal loss and the functional interrelationship between them. It is unclear whether neuronal hypertrophy could be a compensation mechanism for a putative neuronal loss during the diabetes mellitus. (C) 2007 Elsevier Ltd. All rights reserved.

Melatonin and the time window for the expression of the alpha 8 nicotinic acetylcholine receptor in the membrane of chick retinal cells in culture

We have previously shown that melatonin influences the development of alpha 8 nicotinic acetylcholine receptor (nAChR) by measurement of the acetylcholine-induced increase in the extracellular acidification rate (ECAR) in chick retinal cell cultures. Cellular differentiation that takes place between DIV (days in vitro) 4 and DIV 5 yields cells expressing alpha 8 nAChR and results in a significant increase in the ECAR acetylcholine-induced. Blocking melatonin receptors with luzindole for 48 h suppresses the development of functional alpha 8 nAChR. Here we investigated the time window for the effect of melatonin on retinal cell development in culture, and whether this effect was dependent on an increase in the expression of alpha 8 nAChR. First, we confirmed that luzindole was inhibiting the effects of endogenous melatonin, since it increases 2-[(125)I] iodomelatonin (23 pM) binding sites density in a time-dependent manner. Then we observed that acute (15, 60 min, or 12 h) luzindole treatment did not impair acetylcholine-induced increase in the ECAR mediated by activation of alpha 8 nAChR at DIV 5, while chronic treatment (from DIV 3 or DIV 4 till DIV 5, or DIV 3.5 till DIV 4.5) led to a time-dependent reduction of the increase in the acetylcholine-induced ECAR. The binding parameters for [(125)I]-alpha-bungarotoxin (10 nM) sites in membrane were unaffected by melatonin suppression that started at DIV 3. Thus, melatonin surges in the time window that occurs at the final stages of chick retinal cell differentiation in culture is essential for development of the cells expressing alpha 8 nAChR subtype in full functional form. (C) 2010 ISDN. Published by Elsevier Ltd. All rights reserved.

Immunocytochemical characterization of the pregeniculate nucleus and distribution of retinal and neuropeptide Y terminals in the suprachiasmatic nucleus of the Cebus monkey

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are modulated by photic and non-photic stimuli. In rodents, direct photic stimuli reach the SCN mainly through the retinohypothalamic tract (RHT), whereas indirect photic stimuli are mainly conveyed by the geniculohypothalamic tract (GHT). In rodents, retinal cells form a pathway that reaches the intergeniculate leaflet (IGL) where they establish synapses with neurons that express neuropeptide Y (NPY), hence forming the GHT projecting to the SCN. In contrast to the RHT, which has been well described in primates, data regarding the presence or absence of the IGL and GHT in primates are contradictory. Some studies have suggested that an area of the pregeniculate nucleus (PGN) of primates might be homologous to the IGL of rodents, but additional anatomical and functional studies on primate species are necessary to confirm this hypothesis. Therefore, this study investigated the main histochemical characteristics of the PGN and the possible existence of the GHT in the SCN of the primate Cebus, comparing the distribution of NPY immunoreactivity, serotonin (5-HT) immunoreactivity and retinal terminal fibers in these two structures. The results show that a collection of cell bodies containing NPY and serotonergic immunoreactivity and retinal innervations are present within a zone that might be homologous to the IGL of rodents. The SCN also receives dense retinal innervations and we observed an atypical distribution of NPY- and 5-HT-immunoreactive fibers without regionalization in the ventral part of the nucleus as described for other species. These data may reflect morphological differences in the structures involved in the regulation of circadian rhythms among species and support the hypothesis that the GHT is present in some higher primates (diurnal animals). (C) 2009 Elsevier B.V. All rights reserved.

TRPV1 receptors modulate retinal development

We investigated the possible participation of TRPV1 channels in retinal apoptosis and overall development. Retinas from newborn, male albino rats were treated in vitro with capsazepine, a TRPV1 antagonist. The expression of cell cycle markers was not changed after TRPV1 blockade, whereas capsazepine reduced the number of apoptotic cells throughout the retina,increased ERK1/2 and p38 phosphorylation and slightly reduced JNK phosphorylation. The expression of BAD, Bcl-2, as well as integral and cleaved capsase-3 were similar in all experimental conditions. Newborn rats were kept for 2 months after receiving high doses of capsazepine. In their retinas, calbindin and parvalbumin protein levels were upregulated, but only the number of amacrine-like, parvalbumin-positive cells was increased. The numbers of calretinin, calbindin, ChAT, vimentin, PKC-alpha and GABA-positive cells were similar in both conditions. Protein expression of synapsin Ib was also increased in the retinas of capsazepine-treated rats. Calretinin, vimentin, GFAP, synapsin Ia, synaptophysin and light neurofilament protein levels were not changed when compared to control values. Our results indicate that TRPV1 channels play a role in the control of the early apoptosis that occur during retinal development, which might be dependent on MAPK signaling. Moreover, it seems that TRPV1 function might be important for neuronal and synaptic maturation in the retina. (C) 2011 ISDN. Published by Elsevier Ltd. All rights reserved.

Immunocytochemical characterization of the pregeniculate nucleus and distribution of retinal and neuropeptide Y terminals in the suprachiasmatic nucleus of the Cebus monkey

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Macro- and microstructure of the superior cervical ganglion in dogs, cats and horses during maturation

The superior cervical ganglion (SCG) provides sympathetic input to the head and neck, its relation with mandible, submandibular glands, eyes (second and third order control) and pineal gland being demonstrated in laboratory animals. In addition, the SCG's role in some neuropathies can be clearly seen in Horner's syndrome. In spite of several studies published involving rats and mice, there is little morphological descriptive and comparative data of SCG from large mammals. Thus, we investigated the SCG's macro- and microstructural organization in medium (dogs and cats) and large animals (horses) during a very specific period of the post-natal development, namely maturation (from young to adults). The SCG of dogs, cats and horses were spindle shaped and located deeply into the bifurcation of the common carotid artery, close to the distal vagus ganglion and more related to the internal carotid artery in dogs and horses, and to the occipital artery in cats. As to macromorphometrical data, that is ganglion length, there was a 23.6% increase from young to adult dogs, a 1.8% increase from young to adult cats and finally a 34% increase from young to adult horses. Histologically, the SCG's microstructure was quite similar between young and adult animals and among the 3 species. The SCG was divided into distinct compartments (ganglion units) by capsular septa of connective tissue. Inside each ganglion unit the most prominent cellular elements were ganglion neurons, glial cells and small intensely fluorescent cells, comprising the ganglion's morphological triad. Given this morphological arrangement, that is a summation of all ganglion units, SCG from dogs, cats and horses are better characterized as a ganglion complex rather than following the classical ganglion concept. During maturation (from young to adults) there was a 32.7% increase in the SCG's connective capsule in dogs, a 25.8% increase in cats and a 33.2% increase in horses. There was an age-related increase in the neuronal profile size in the SCG from young to adult animals, that is a 1.6-fold, 1.9-fold and 1.6-fold increase in dogs, cats and horses, respectively. on the other hand, there was an age-related decrease in the nuclear profile size of SCG neurons from young to adult animals (0.9-fold, 0.7-fold and 0.8-fold in dogs, cats and horses, respectively). Ganglion connective capsule is composed of 2 or 3 layers of collagen fibres in juxtaposition and, as observed in light microscopy and independently of the animal's age, ganglion neurons were organised in ganglionic units containing the same morphological triad seen in light microscopy. Copyright (c) 2007 S. Karger AG, Basel.

Alterations in the fat body cells of rhinocricus padbergi (Diplopoda) resulting from exposure to substrate containing sewage sludge

Abstract The final disposal of residues generated at sewage treatment plants (STPs) has become a major problem for cities, due to the increase in the amount of treated sewage. One of the alternatives for the residue, labeled sewage sludge, is its reuse in agriculture and in degraded soil. However, not all pathogens and metals present in it are eliminated during treatment. Diplopods have been used as bioindicators in ecotoxicological tests as they are constantly in close contact with the soil. Owing to this fact, the purpose of this study was to expose specimens of the diplopod Rhinocricus padbergi to substrate containing sewage sludge collected at STPs to analyze morphological alterations in their parietal and perivisceral fat body, where substances are stored. The exposures were held for 7, 15, or 90 days at different concentrations of sewage sludge (control, 1%, 10%, and 50%). The parietal fat body showed no alterations in any of the three exposure periods or concentrations. Alterations in the perivisceral fat body were observed for all exposure periods. According to the results, we suggest that the sludge used has toxic agents responsible for changing the animal's perivisceral fat body. © 2012 Microscopy Society of America.

Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells

The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5), while the Bmax value for 125I-a-bungarotoxin was reduced. Despite the presence of a8-like immunoreactivity at DIV4, functional responses mediated by a-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 µM) was essentially without effect at DIV4 and DIV5, while dihydro-ß-erythroidine (10-100 µM) blocked the response to acetylcholine (3.0 nM-2.0 µM) only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the a-bungarotoxin-sensitive response at DIV5. Therefore, a-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an a-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by a-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express a-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.

Pyrostegia venusta heptane extract containing saturated aliphatic hydrocarbons induces apoptosis on B16F10-Nex2 melanoma cells and displays antitumor activity in vivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In Vitro Effects of Bevacizumab Treatment on Newborn Rat Retinal Cell Proliferation, Death, and Differentiation

PURPOSE. Vascular endothelial growth factor (VEGF) is an important signal protein in vertebrate nervous development, promoting neurogenesis, neuronal patterning, and glial cell growth. Bevacizumab, an anti-VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on cell death, proliferation, and differentiation in newborn rat retina. METHODS. Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA, to detect cell proliferation); caspase-3 and beclin-1 (to investigate cell death); and vimentin and glial fibrillary acidic protein (GFAP, markers of glial cells). Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. Results from treatment and control groups were compared. RESULTS. No significant difference in the staining intensity (on immunohistochemistry) of PCNA, caspase-3, beclin-1, and GFAP, or in the levels of PCNA, caspase-3, beclin-1, and vimentin mRNA was observed between the groups. However, a significant increase in vimentin levels and a significant decrease in GFAP mRNA expression were observed in bevacizumab-treated retinal explants compared with controls. CONCLUSIONS. Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation by increasing vimentin levels and downregulating GFAP gene expression. Thus, we suggest anti-VEGF agents be used with caution in developing retinal tissue. (Invest Ophthalmol Vis Sci. 2012;53:7904-7911) DOI:10.1167/iovs.12-10283

Tissue response and wound healing after placement of two types of bioengineered grafts containing vital cells in submucosal maxillary pouches: an experimental pilot study in rabbits

PURPOSE: This pilot study evaluated the wound healing and tissue response after placement of two different skin substitutes in subgingival mucosal pouches in rabbits. MATERIALS AND METHODS: Four rabbits were selected to receive a commercially available skin substitute consisting of a collagen matrix with fibroblasts and an epithelial layer (test membrane 1) and a prototype device consisting of a collagen matrix with fibroblasts only (test membrane 2). In each rabbit, two horizontal incisions were made in the buccal alveolar mucosa of the maxilla bilaterally to create submucosal pouches. Three pouches in each animal were filled with either the test 1 or test 2 membranes, and one pouch was left without a membrane (sham-operated control). All rabbits were sacrificed after a healing period of 4 weeks, and histologic samples were prepared and examined. RESULTS: After a healing period of 1 month, both tested membranes were still visible in the sections. Test membrane 1 was still bilayered, contained inflammatory cells in its center, and was encapsulated by a thick fibrous tissue. Numerous ectopic calcifications were evident in the collagenous part of the membrane and in association with some basal epithelial cells. Test membrane 2 was also encapsulated in fibrous tissue, with inflammatory cells present only between the fibrous encapsulation and the remnants of the membrane. For test membrane 2, no calcifications were visible. CONCLUSIONS: Test membrane 1 seemed to be more resistant to degradation, but there was also a more pronounced inflammatory reaction in comparison to test membrane 2, especially in the vicinity of the keratinocytes. The significance of the ectopic calcifications, along with that of the resorption or degradation processes of both tested membranes, must be evaluated in future experimental studies, with different time points after implantation examine