Transferrin secretion in medium of Müller glial cells from 8 to 12-day-old wild-type and transgenic mice decreases with the subcultures

A: Mouse transferrin (mTf) secretion in medium of cultured Müller glial (MG) cells from wild-type ( WT) and transgenic (Tg) mice at confluency after primary culture (P0) and at the first (P1) and the second passages (P2) was measured by radioimmunoassay (RIA; ng/ml). Each column represents the mean ±SEM. The asterisk represents statistical significance of differences between WT and Tg at P0, p<p><b>Copyright information:</b></p><p>Taken from "The protective role of transferrin in Müller glial cells after iron-induced toxicity"</p><p></p><p>Molecular Vision 2008;14():928-941.</p><p>Published online 20 May 2008</p><p>PMCID:PMC2391081.</p><p></p

Müller glial cells from transgenic mice are more resistant to iron-mediated stress than Müller glial cells from wild-type mice

A: Culture of Müller glial (MG) cells from wild-type (WT) and transgenic (Tg) mice at the first passage were treated with 100 µM of FeCl-NTA (FN), during 96 h. The number of cells was evaluated by counting in comparison to control condition. Each column represents the mean ±SEM. The triple asterisk represent statistical significance of differences between treated and control, respectively, for WT and Tg, and between control and treated, p<p><b>Copyright information:</b></p><p>Taken from "The protective role of transferrin in Müller glial cells after iron-induced toxicity"</p><p></p><p>Molecular Vision 2008;14():928-941.</p><p>Published online 20 May 2008</p><p>PMCID:PMC2391081.</p><p></p

Mouse transferrin and human transferrin proteins expression are modulated after iron stress

A: Mouse transferrin (mTf) secretion was measured by radioimmunoassay (RIA) in the culture medium of Müller glial (MG) cells from wild-type (WT) and transgenic (Tg) mice in control condition or after addition of 100 µM of FeCl-NTA (FN) during 96 h. Each column represents the mean ±SEM. The double asterisk represents statistical significance of differences between treated and control, respectively, for WT and Tg, p<p><b>Copyright information:</b></p><p>Taken from "The protective role of transferrin in Müller glial cells after iron-induced toxicity"</p><p></p><p>Molecular Vision 2008;14():928-941.</p><p>Published online 20 May 2008</p><p>PMCID:PMC2391081.</p><p></p

<i>In-vivo</i> PKCζ inhibition in 6-month-old-diabetic rats partially restores PKCζ staining pattern in cone outer segments.

<p>The phosphorylated active form PKCζ-P (green) was found in IS of both control (CTL) and diabetic (DIA) rats (a). Diabetic inhibitor (DIA+IZ) treated rats showed partial restoration of OS PKCζ (red) staining (a). We performed laser micro dissection of the photoreceptor layer on cryosections (b) to study layer-specific protein level by Western blotting analysis. The PKCζ-P immunoreactivity increase found in diabetic rats (DIA) was partially restored in treated rats (DIA+IZ).</p

Immunolocalization of PKCζ and occludin in retinal pigment epithelium (RPE) flatmounts.

<p>In 6-month-old controls, PKCζ staining (red) appeared as focal spots within the loops depicted by occludin (green) protrusions (a). In age-matched diabetic conditions, no PKCζ labeling (box and green arrow) was observed (b). After 12 month of diabetes marked disruption of intercellular junctions (arrows) was evidenced (c). At this stage the PKCζ at the junctions (TJ)/total amount of PKCζ ratio was significantly decreased for diabetic conditions (Image J software, National Institutes of Health, Bethesda, MD) (d) and occludin internalization from the cell membrane into the cytoplasm was found (e). The colocalization of PKCζ (red) and PKCζ-P (green) found in 12-month-old controls (f) was not present in age-matched diabetics (g). Furthermore the activated PKCζ-P form exhibited a discontinuous staining along the cell membrane (g).</p

Diabetes destabilizes and down regulates PAR3/PAR6 PKCζ-associated protein complex.

<p>a) Upper panels: In 12-month-old diabetic rats (DIA) PAR6 distribution in flatmount RPE showed a clear cytoplasmic relocation of PAR6 compared to age-matched control rats (CTL) where PAR6 appeared as relative regular punctiform staining at the TJ levels. The same applied to PAR3 staining distribution (Lower panels). b) PAR6 and PAR3 immunoblotting on RPE cell extracts from 12-month-old rats showed a significant decrease of both protein levels in diabetic conditions (statistical analysis was performed on the PAR3 180 KDa isoform).</p

Alteration of PKCζ distribution in the outer retina is associated with cone outer segment and OLM disruption in diabetic rats.

<p>PKCζ staining was located in inner segments (IS) of rod and cones and exclusively in cone OS as evidenced by PKCζ-PNA double labeling in 6-month-old control rats (a). In 6-month-old diabetic rats, no OS staining was detected (a). Furthermore PKCζ staining (red) was lost in S-cones, specifically marked by Blue opsin staining (green), as compared to controls. Some S-cone OS also showed marked structural alterations, suggesting early photoreceptor degeneration (b). In 12-month-old diabetic rats OLM discontinuity (arrows) was evidenced (c). OLM tight-junction disruption was further confirmed on retinal flatmounts by an occludin/PKCζ double staining (d, white arrows).</p

Diabetes stage-specific retinal pigment epithelium (RPE) level of PKCζ and its activated phosphorylated form PKCζ-P T410.

<p>No significant changes were observed for PKCζ level at any stage between control and diabetic conditions. To the contrary, PKCζ-P T410 immunoreactivity was significantly (*P<0.05, Mann–Whitney test) increased (by around 40%) at 6 months and then decreased (by around 60%) at 12 months of diabetes compared to controls.</p

Diabetes induces outer retinal edema and loss of cones photoreceptors.

<p>Diabetic retina (DIA) demonstrated increased extracellular spaces within the outer nuclear layer (ONL) and photoreceptor segment disorganization, consistent with an edematous aspect of the outer retina (Historesin (a) and semithin sections (b)). Peanut agglutinin labeling, a specific marker of cone extracellular matrix, evidenced a marked decreased in cell cone density (c). Whole retina quantification of cones evaluated the net loss to be around 20% (d) as compared to controls (p = 0.002, Mann–Whitney test).</p

Characteristics of the study animals.

<p>a: diabetes versus controls, P value<0.05.</p