Neuronal damage does not differ between left and right hemispheres in the CA1 region of the hippocampus.

<p><b>A</b>. Representative hematoxylin and eosin-stained tissue sections show pyknotic neurons (black arrowheads) in the CA1 regions of the hippocampi obtained from controls and P1VEH rats. Sections of the CA1 regions of the left and right hippocampi regions from control rats show an abundance of normal neurons (white arrowheads). <b>B</b>. Quantitative analysis reveals a significantly higher number of pyknotic neurons in vehicle rats compared to corresponding control animals. There are no differences in pyknotic neuronal cell count between left and right hippocampi regions of P1VEH rats. Scale bars  = 10 µm. Error bars indicate S.D. *P<0.05 determined using Student's <i>t</i> test.</p

Immunocytochemical analysis of iOPN expression.

<p><b>A</b>: Representative sections exhibiting iOPN expression from the left (top) and right (bottom) cortex in control, P1VEH and P1ACET animals. We detected iOPN in the cytoplasm (labeled C, insets). A negative staining control from the right cortex is shown for comparison. In the right cortex, higher iOPN expression was evident in vehicle rats compared to control and ACET (15 mg/kg)-treated rats. <b>B</b>. The intensity of staining was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014568#s2" target="_blank">Methods</a>. The right cortex of vehicle rats shows a significant increase in iOPN expression compared to ACET (15 mg/kg)-treated rats. *P<0.05 determined using one-way ANOVA followed by Tukey's post test. Error bars indicate S.D. Scale bars  = 25 µm.</p

Schematic of experimental timeline.

<p>Rats were surgically instrumented and maintained under steady-state conditions for 15 min prior to initiating I-R. At that point, animals were rendered hypotensive (mean arterial pressure ∼55 mm Hg) by withdrawing 5 mL of blood, and the carotid arteries were clamped to induce ischemia. Reperfusion was begun by unclamping of the carotid arteries and returning the withdrawn blood to the animal. Control animals were surgically instrumented but were not subject to hypotension or I-R. In Protocol 1, acetaminophen (ACET) (15 mg/kg) was only administered prior to ischemia, whereas in Protocol 2, ACET (15 mg/kg) was administered prior to ischemia and again immediately before the onset of reperfusion.</p

iOPN expression in the hippocampus, left cortex and right cortex.

<p><b>A</b>. Representative Western blots of cytosolic fractions from the hippocampus, left cortex and right cortex of control, P1VEH, and P1ACET rats reveal a ∼30 kDa OPN species; species of lower mobility were not detected. Control animals were instrumented, but not subject to I-R. <b>B</b>. Quantitative densitometric analysis of iOPN bands normalized to their corresponding β-tubulin bands confirmed a significant increase of iOPN in vehicle rats in the right cortex. <b>C</b>. Representative Western blots of cytosolic fractions from the right cortex of control, P2VEH and P2ACET rats. <b>D</b>. Quantitative densitometric analysis of iOPN bands normalized to their corresponding β-tubulin bands confirmed a significant increase in iOPN expression in P2VEH rats that is reduced by about 50% in the P2ACET rats. Error bars indicate S.D. *P<0.05 determined using one-way ANOVA followed by Tukey's post test.</p

Acetaminophen (ACET) maintains mitochondrial cytochrome c content in rats subjected to Protocol 2.

<p><b>A</b>: Representative Western blots of the mitochondrial fractions from the right cortex of control, P2VEH, and P2ACET rats. <b>B</b>: Quantitative densitometric analysis of cytochrome c bands normalized to their corresponding VDAC bands confirms a significant decrease in cytochrome c content in vehicle rats. Error bars indicate S.D. *P<0.001 determined using Student's <i>t</i> test.</p

Elevated Nrf-2 responses are insufficient to mitigate protein carbonylation in hepatospecific PTEN deletion mice

<div><p>Objective</p><p>In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease (NASH) is oxidative stress, which leads to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine the impact of NASH on protein carbonylation and antioxidant responses in a murine model.</p><p>Methods</p><p>Liver-specific phosphatase and tensin homolog (PTEN)-deletion mice (PTEN^LKO) or control littermates were fed a standard chow diet for 45–55 weeks followed by analysis for liver injury, oxidative stress and inflammation.</p><p>Results</p><p>Histology and Picrosirius red-staining of collagen deposition within the extracellular matrix revealed extensive steatosis and fibrosis in the PTEN^LKO mice but no steatosis or fibrosis in controls. Increased steatosis and fibrosis corresponded with significant increases in inflammation. PTEN-deficient livers showed significantly increased cell-specific oxidative damage, as detected by 4-hydroxy-2-nonenal (4-HNE) and acrolein staining. Elevated staining correlated with an increase in nuclear DNA repair foci (γH2A.X) and cellular proliferation index (Ki67) within zones 1 and 3, indicating oxidative damage was zonally restricted and was associated with increased DNA damage and cell proliferation. Immunoblots showed that total levels of antioxidant response proteins induced by nuclear factor erythroid-2-like-2 (Nrf2), including GSTμ, GSTπ and CBR1/3, but not HO-1, were elevated in PTEN^LKO as compared to controls, and IHC showed this response also occurred only in zones 1 and 3. Furthermore, an analysis of autophagy markers revealed significant elevation of p62 and LC3II expression. Mass spectrometric (MS) analysis identified significantly more carbonylated proteins in whole cell extracts prepared from PTEN^LKO mice (966) as compared to controls (809). Pathway analyses of identified proteins did not uncover specific pathways that were preferentially carbonylated in PTEN^LKO livers but, did reveal specific strongly increased carbonylation of thioredoxin reductase and of glutathione-<i>S</i>-transferases (GST) M6, O1, and O2.</p><p>Conclusions</p><p>Results show that disruption of PTEN resulted in steatohepatitis, fibrosis and caused hepatic induction of the Nrf2-dependent antioxidant system at least in part due to elevation of p62. This response was both cell-type and zone specific. However, these responses were insufficient to mitigate the accumulation of products of lipid peroxidation.</p></div

Impact of PTEN^LKO on expression of antioxidant responses.

<p>Western immunoblotting analysis of GCLC, GSTμ, GSTπ, CBR1/3, HO-1, Prdx5 and Gpx1 in liver lysates prepared from control and PTEN^LKO mice. All exposures were normalized using GAPDH expression. Data are means +/- SEM, n = 6 per genotype.</p

Basic pathology in PTEN^LKO livers.

<p>Panels A, E. Histology of livers from control or PTEN^LKO mice. Panels B, F. Bright-field images of PSR-staining. Panels C, G. Polarized light exposure of PSR-staining. Panels D, H. Cytokeratin 7 staining. Representative images are shown; n = at least 3 mice per genotype (CV, central vein, PT, portal triad).</p

Impact of PTEN^LKO on protein carbonylation.

<p>Tissue sections isolated from control and PTEN^LKO mice were immunostained using antibodies directed against 4-HNE or acrolein, as indicated. A. 100X magnification, B. 400X magnification of PTEN^LKO livers. Representative images are shown; n = at least 3 mice per genotype. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198139#pone.0198139.g001" target="_blank">Fig 1</a>.</p

Inflammatory infiltrates in PTEN^LKO livers.

<p>Liver sections from control and PTEN^LKO mice were immunostained using antibodies directed against MPO (panels A, E); F4/80 (panels B, F); CD3 (panels C, G); or B220 (panels D, H). Representative images are shown; n = at least 3 mice per genotype. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198139#pone.0198139.g001" target="_blank">Fig 1</a>.</p