Time course modifications in organotypic culture of human neuroretina

The purpose of this study was to characterize organ culture of human neuroretina and to establish survival and early degeneration patterns of neural and glial cells. Sixteen neuroretina explants were prepared from 2 postmortem eyes of 2 individuals. Four explants were used as fresh retina controls, and 12 were evaluated at 3, 6, and 9 days of culture. Neuroretina explants (5 × 5 mm) were cultured in Transwell® dishes with the photoreceptor layer facing the supporting membrane. Culture medium (Neurobasal A-based) was maintained in contact with the membrane beneath the explant. Cryostat and ultrathin sections were prepared for immunohistochemistry and electron microscopy. Neuroretinal modifications were evaluated after toluidine blue staining and after immunostaining for neuronal and glial cell markers. Ultrastructural changes were analyzed by electron microscopy. From 0 to 9 days in culture, there was progressive retinal degeneration, including early pyknosis of photoreceptor nuclei, cellular vacuolization in the ganglion cell layer, decrease of both plexiform layer thicknesses, disruption and truncation of photoreceptor outer segments (OS), and marked reduction in the number of nuclei at both nuclear layers where the cells were less densely packed. At 3 days there was swelling of cone OS with impairment of pedicles, loss of axons and dendrites of horizontal and rod bipolar cells that stained for calbindin (CB) and protein kinase C (PKC-α), respectively. After 9 days, horizontal cells were pyknotic and without terminal tips. There were similar degenerative processes in the outer plexiform layer for rod bipolar cells and loss of axon terminal lateral varicosities in the inner plexiform layer. Glial fibrillary acidic protein (GFAP) staining did not reveal a dramatic increase of gliosis in Müller cells. However, some Müller cells were CB immunoreactive at 6 days of culture. Over 9 days of culture, human neuroretina explants underwent morphological changes in photoreceptors, particularly the OS and axon terminals, and in postsynaptic horizontal and bipolar cells. These early changes, not previously described in cultured human samples, reproduce some celullar modifications after retinal damage. Thus, this model may be suitable to evaluate therapeutic agents during retinal degeneration processes.

Age-related functional and structural retinal modifications in the Igf1−/− null mouse

Background. Mutations in the gene encoding human insulin-like growth factor-I (IGF-I) cause syndromic neurosensorial deafness. To understand the precise role of IGF-I in retinal physiology, we have studied the morphology and electrophysiology of the retina of the Igf1−/− mice in comparison with that of the Igf1+/− and Igf1+/+ animals during aging. Methods. Serological concentrations of IGF-I, glycemia and body weight were determined in Igf1+/+, Igf1+/− and Igf1−/− mice at different times up to 360 days of age. We have analyzed hearing by recording the auditory brainstem responses (ABR), the retinal function by electroretinographic (ERG) responses and the retinal morphology by immunohistochemical labeling on retinal preparations at different ages. Results. IGF-I levels are gradually reduced with aging in the mouse. Deaf Igf1−/− mice had an almost flat scotopic ERG response and a photopic ERG response of very small amplitude at postnatal age 360 days (P360). At the same age, Igf1+/− mice still showed both scotopic and photopic ERG responses, but a significant decrease in the ERG wave amplitudes was observed when compared with those of Igf1+/+ mice. Immunohistochemical analysis showed that P360 Igf1−/− mice suffered important structural modifications in the first synapse of the retinal pathway, that affected mainly the postsynaptic processes from horizontal and bipolar cells. A decrease in bassoon and synaptophysin staining in both rod and cone synaptic terminals suggested a reduced photoreceptor output to the inner retina. Retinal morphology of the P360 Igf1+/− mice showed only small alterations in the horizontal and bipolar cell processes, when compared with Igf1+/+ mice of matched age. Conclusions. In the mouse, IGF-I deficit causes an age-related visual loss, besides a congenital deafness. The present results support the use of the Igf1−/− mouse as a new model for the study of human syndromic deaf-blindness.

Polyaniline/Montmorillonite Nanocomposites Obtained by In Situ Intercalation and Oxidative Polymerization in Cationic Modified-Clay (Sodium, Copper and Iron)

Polyaniline/montmorillonite nanocomposites (PANI/M) were obtained by intercalation of aniline monomer into M modified with different cations and subsequent oxidative polymerization of the aniline. The modified-clay was prepared by ion exchange of sodium, copper and iron cations in the clay (Na–M, Cu–M and Fe–M respectively). Infrared spectroscopy confirms the electrostatic interaction between the oxidized PANI and the negatively charged surface of the clay. X-ray diffraction analysis provides structural information of the prepared materials. The nanocomposites were characterized by transmission electron microscopy and their thermal degradation was investigated by thermogravimetric analysis. The weight loss suggests that the PANI chains in the nanocomposites have higher thermal stability than pure PANI. The electrical conductivity of the nanocomposites increased between 12 and 24 times with respect to the pure M and this increase was dependent on the cation-modification. The electrochemical behavior of the polymers extracted from the nanocomposites was studied by cyclic voltammetry and a good electrochemical response was observed.

Metal-complex ionosilicas: Cationic mesoporus silica with Ni(II) and Cu(II) complexes in their framework

Metal-complex ionosilicas with cationic complexes into the mesoporous silica framework were prepared using anionic surfactants. The electrostatic interaction between the anionic surfactant and the cationic metal complexes incorporated in the silica framework allows for the fine tuning of the mesoporous structure. The gentle procedure of synthesis developed and mild ion-exchange extraction of the surfactant, allowed a cleaner route for the immobilization of homogeneous cationic catalysts in mesoporous silica, while protecting the structural and chemical integrity of the metal complexes.