Biomedical subjects

Alexandre Hiroaki Kihara

Publications and source records attributed to Alexandre Hiroaki Kihara.

5 recordsLinked to original sources

PubMed2006

Prolonged dark adaptation changes connexin expression in the mouse retina.

In the retina, ambient light levels influence the cell coupling provided by gap junction (GJ) channels, to compensate the visual function for various lighting conditions. However, the effects of ambient light levels on expression of connexins (Cx), the proteins that form the GJ channels, are poorly understood. In the present study, we first determined whether gene expression of specific Cx (Cx26, Cx31.1, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, and Cx57) was affected by prolonged dark adaptation. Cx mRNA relative levels were determined in mouse retinas dark adapted for 3 hr, 1 day, and 7 days by using quantitative real-time PCR. Transcript levels of some Cx were repressed after 3 hr (Cx57), 1 day (Cx45), or 7 days (Cx36 and Cx43) of dark adaptation; others were increased after 1 day (Cx50) or 7 days (Cx31.1 and Cx37); and two of them (Cx26 and Cx40) were not significantly altered. The second aim was to determine whether prolonged dark adaptation affects protein expression of two important Cx in retina: neuronal Cx36 and glial Cx43. We were able to demonstrate that important changes in protein distribution and expression also took place in retina during long-term dark adaptation. Given their localization, the specific alterations in Cx expression may reflect their distinct response to ambient light levels.

Animals↗

PubMed2006

Small GTP-binding protein RhoB is expressed in glial Müller cells in the vertebrate retina.

Among several small Rho GTPases observed in the chick retina, RhoB was transiently expressed during development and mainly present in glial Müller cells in the adult. The aim of this study was to compare the distribution of RhoB in the chick and mouse adult retinas and to study its potential role in the maintenance of cell morphology. The distribution of RhoB was studied in situ and pure Müller cell cultures were submitted to Clostridium difficile toxin A and lysophosphatidic acid (LPA) treatment in order to inactivate and activate Rho proteins, respectively. Cell morphology, F-actin arrangement, RhoB, and vimentin distribution were studied by immunofluorescence and confocal microscopy. The results showed that, in both species, all vimentin-containing cells also expressed RhoB in situ and in vitro. Toxin A promoted cell rounding and detachment due to actin depolymerization, changing the distribution of RhoB only in chick cells. In serum-starved cells LPA stimulated actin polymerization and cell spreading, but only in chick cells was RhoB distribution recruited to expanding cellular processes and newly formed focal adhesions. These data suggest that, although RhoB is expressed by Müller cells in chick and mouse, its role in the maintenance of cellular morphology and regulation may be different. In addition, we show that RhoB may be an interesting Müller cell marker in the adult retina.

Animals↗

PubMed2006

Expression of connexins 36, 43, and 45 during postnatal development of the mouse retina.

Gap junction channels formed by connexins (Cx) may play essential roles in some processes that occur during retinal development, such as apoptosis and calcium wave spread. The present study was undertaken to determine the distribution pattern of Cx36, Cx43, and Cx45 by immunofluorescence, as well as their gene expression levels by quantitative PCR during postnatal development of the mouse retina. Our results showed an increased expression of neuronal Cx36 from P1 until P10, when this Cx reached adult levels, and it was mainly distributed in the outer and inner plexiform layers. In turn, Cx43 was almost absent in retinal progenitor cells at P1, it became more prominent in glial cell processes about P10, and did not change until adulthood. Double-labeling studies in situ and in vitro with antivimentin, a Müller cell marker, confirmed that Cx43 was expressed by these cells. In addition, quantitative PCR showed that Cx43 and vimentin shared very similar temporal expression patterns. Finally, in contrast to Cx36 and Cx43, Cx45 mRNA was strongly down-regulated during development. In early postnatal days, Cx45 was seen ubiquitously distributed throughout the retina in cells undergoing proliferation and differentiation, as well in differentiated neurons. In adult retina, this protein had a more restricted distribution both in neurons and glial cells, as confirmed in situ and in vitro. In conclusion, we observed a distinct temporal expression pattern for Cx36, Cx43, and Cx45, which is probably related to particular roles in retinal function and maintenance of homeostasis during development of the mouse retina.

Age Factors↗

PubMed2005

Effects of ambient lighting on visual discrimination, forward masking and attentional facilitation.

Differences between neural processing underling day and night vision occur at initial transduction steps. However, comparison of a visual task performance in photopic versus scotopic situations has an intrinsic problem: ambient light levels directly affect the contrast between stimuli and background. By using a simple but innovative method, we were able to maintain the stimuli/background contrast in order to appropriately evaluate the effects of ambient lighting on visual discrimination, attentional facilitation and forward masking. Our results revealed that ambient light levels does not affect spatial accuracy in central vision, but peripheral stimuli are more rapidly recognized when presented in photopic conditions. Additionally, our data suggest that ambient lighting do not unbalance the opposing effects of attentional facilitation versus forward masking. Modulation of cell coupling that takes place in the retina triggered by ambient light levels may underlie differences in visual discrimination in photopic and scotopic conditions.

Adult↗

PubMed2005

Protecting RNA in fixed tissue: an alternative method for LCM users.

RNA degradation is a major drawback in most common fixation protocols in techniques that require both RNA integrity and preserved morphology, such as laser capture microdissection (LCM) followed by RT-PCR. Moreover, RNA isolation kits especially developed for LCM samples are very expensive. Our aim was to determine an easy protocol that ideally must provide an acceptable morphology, allow proper laser capture of selected cells and improve RNA yield and quality. In this study, retinas were dissected, briefly incubated in a RNA preservative and fixed in 2% paraformaldehyde before being cut on a cryostat. LCM was carried out in retinal sections for immediate RNA isolation, by using TRIzol common protocol with minor modifications. Real-time PCR was performed next in order to compare availability of RNA from samples submitted to different protocols. The use of the RNA preservative followed by a fast fixation did not jeopardize tissue morphology, allowing microdissection of selected cells, combined to minor modifications in usual RNA isolation procedures, significantly improved RNA yield and quality. Furthermore, only LCM samples submitted to our protocol provided amplifiable mRNA, as determined by real-time PCR. Taken together, the combination of the described procedures resulted in a reliable alternative for LCM users.

Animals↗