PTEN expression in the CA3 area of the hippocampus.

<p>Immunofluorescence showing PTEN expression in the CA3 area 1 day after kainic acid i.p. injection (B) and in control mice treated only by saline (A). Nuclei were stained with nuclear marker DAPI. Scale bar: 50 µm.</p

Activation of c-Jun after kainate stimulation in the CA3 area of the hippocampus.

<p>The P-c-Jun immunoreactivity, absent in control mice (A), can be observed in neurons of animals treated with kainate (B) or with both kainate and PTEN inhibitor (C), sacrificed one day after the treatment. At higher magnification, nuclei of neurons positive for P-c-Jun after kainate treatment. D. Immunostaining showing that P-c-Jun (red) is revealed in the nucleus of neurons labeled with NeuN (green) in the CA3 area of the hippocampus, one day after the kainate stimulation. Scale bar: 50 µm. E. Histogram showing the % of P-c-Jun staining in hippocampal CA3 subfield one day following KA injection and PTEN inhibition by bpv(pic). As it can be observed, bpv(pic) is unable to prevent the significant increase in the P-c-Jun immunoreactivity caused by kainate treatment. *<i>P</i><0.05.</p

Astrogliosis in the CA3 area of the hippocampus following kainic acid injection.

<p>Glial fibrillary acidic protein (GFAP)-positive profiles in the hippocampi of control (A), kainic acid (KA)-treated, 1 day survival mice (B) and KA/bpv(pic)-treated, 1 day survival mice (C) (scale bar: 50 µm). D. Histogram showing the % of GFAP staining in the hippocampal CA3 area one day after kainic acid injection and PTEN inhibition by bpv(pic). The significant increase in GFAP immunoreactivity after kainate administration, expressed as a percentage of the whole area occupied by positive profiles, is significantly prevented by bpv(pic) treatment. *<i>P</i><0.05, §<0.05.</p

PTEN expression in mitochondrial and cytosolic fraction after the excitotoxic stimulus.

<p>Representative Western blot (A) and relative quantification (B) showing mitochondrial PTEN translocation in control (ctr), kainic treated group (KA) and kainic/bpv(pic) group (KA+I) 3, 6 and 12 h after the kainate treatment. As it can be observed, the excitotoxic stimulus leads to a significant increase in mitochondrial PTEN expression, significantly decreased 6 h after kainic acid injection in animals treated with bpv(pic). On the contrary, in the cytosolic fraction kainic acid or PTEN inhibitor does not lead to significant differences in PTEN levels in any time points considered as it can be observed from representative Western blots (C) and relative quantification (D). Data are expressed as mean ± S.E.M., *<i>P</i><0.05, #<i>P</i><0.05, **<i>P</i><0.01 with Two way ANOVA and Bonferroni post hoc test. Experiments were repeated three times.</p

PTEN inhibition protects CA3 neurons from kainate-induced excitotoxicity.

<p>Nissl-staining of hippocampus sections of mice treated with saline (A, control), with kainate (B, KA) or with both kainate and bpv(pic) (C, KA+I), killed one day after treatment (scale bar: 1 mm). A1. CA3 area of control animals (ctr) treated with saline only (scale bar: 50 µm). B1. CA3 area of animals treated with kainate (KA). Arrowheads point to apoptotic bodies and condensed nuclei as a consequence of kainate treatment. At higher magnification, a neuron that is degenerating. C1. CA3 area of animals treated with both kainate and PTEN inhibitor (KA+I). D. Immunostaining showing cleaved caspase-3 (in red) in neurons labeled with MAP-2 (in green) in the CA3 area of the hippocampus, one day after kainate administration. At higher magnification, arrowheads indicating neurons with a clear expression of cleaved caspase-3. Scale bar: 50 µm (30 µm at higher magnification). E. Surviving neurons in the hippocampal CA3 region. Histogram showing the linear density (cells/mm) of surviving neurons in the CA3 area of the hippocampus one day following kainic (KA) acid injection or kainic (KA) acid injection and PTEN inhibition (I) by bpv(pic) (KA+I). **<i>P</i><0.01, §§<i>P</i><0.01.</p

Effects of 3-MA treatment in I/R after 24 h.

<p>In 3-MA-treated I/R retinas treated, LC3-positivity is markedly reduced (A), whereas lysosomal activity (LAMP1) is unchanged (B) compared to I/R retinas. (C) Cleaved caspase-3-positive cells are absent in the untreated retinas (upper panel), whereas a strong increase in cleaved caspase 3-positivity in the GCL is seen at 24 h (middle panel), significantly prevented by 3-MA treatment (bottom panel). (D) TUNEL-positive neurons are found occasionally in the control retina, whereas they increase dramatically after I/R in the GCL (arrowheads) and in INL (arrow). This increase in TUNEL-positive cells is prevented by 3-MA treatment (bottom panel). (E) I/R increase GFAP immunoreactivity (red) in the retina: in the untreated retina, only the end feet of the Müller cells (arrowheads) are GFAP-positive. GFAP expression strongly increase 24 h after I/R, and is prevented by 3-MA administration. In the I/R and I/R + 3-MA retinas, GFAP positivity is detectable in the end feet (arrowheads) and radial processes (arrows) of Müller cells. Following 3-MA treatment, the immunoreactivity is decreased versus I/R retina especially in the end feet of Müller cells. ILM: inner limiting membrane. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022514#pone-0022514-g001" target="_blank">Figure 1</a>. (*): vitreous. Scale bar: 50 µm (100 µm in D).</p

Immunoblot analysis of LC3 I and II expression in the retina after IOP.

<p>(A) Autoradiography of the western blot probed with anti-LC3 and anti-βIII tubulin antibodies. (B) Quantitation of the image in (A) after normalization with βIII tubulin. Level of LC3-II in retinas increase by 20% 24 hours after IOP compared with the control (n = 2, *P<0.05). LC3-I expression in the retina does not change significant after IOP (data not shown in B).</p

Experimental plan.

1<p>For each animal, the left eye was injected, the right eye served as control.</p>2<p>IHC was performed on 2 animals of each group.</p

Immunofluorescence against LAMP1 and LC3.

<p>All sections were counterstained with bisbenzimide (Hoechst staining) to show retinal layers. Twelve hours after I/R, LAMP1 positive cytoplasmic granules are present (A) and 24 h after I/R both LAMP1 (B) and LC3 (C) positive granules are present in the GCL (arrow) and INL (arrowheads). LC3-positive vesicles are more represented than LAMP1 vesicles. LC3-immunopositivity is absent after 48 h (D). At high magnification (E–F), clear cytoplasmic lysosomal LAMP1 positive vesicles (arrowheads) can be observed in the GCL at 24 h from the insult (E). LC3 labelling appear as numerous fluorescent dots (arrowheads) after 24 h from the I/R (F). Double immunolabeling against LAMP1 (red) and LC3 (green) at 24 h shows the relationship between autophagosomes and lysosomes (G). The increase in punctuate LC3 and LAMP1 occurs in the same neurons after 24 h (G, arrowheads). At high magnification, confocal microscopy reveals that autophagic marker and LAMP1 colocalize, suggesting that fusion of autophagosomes with lysosomes occur in dying neurons after I/R (arrowheads, H). Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022514#pone-0022514-g001" target="_blank">Figure 1</a>. Scale bars = 50 µm (A); 100 µm (B, C, D and G) and 5 µm (E, F and H).</p

Cell counts in the GCL.

<p>Representative transverse sections through rat retina at 24 h, Hoechst staining. Retinal thickness is markedly reduced, compared to the control (A), after injury (B), mainly due to decrease in IPL thickness and number of GCL-neurons; these effects are partially prevented by 3-MA treatment (C). No statistical significant alterations are apparent in the other layers. (D) Quantification of GCL-neurons numbers in each group: in the I/R retina, the number of the GCL-neurons significantly decrease compared to controls (** <i>P<0.01</i>); 3-MA partially prevent neuron death (* <i>P<0.05</i>). Scale bar: 50 µm.</p