Mild carotid stenosis creates gradual, progressive, lifelong brain, and eye damage: An experimental laboratory rat model.

In humans, carotid stenosis of 70% and above might be the cause of clinical symptoms such as transient ischemic attack and stroke. No clinical or animal studies have evaluated mild carotid occlusion, and few examined unilateral occlusion. Here, Westar rats underwent bilateral or unilateral carotid occlusion of 28-45%. Long-term effects were evaluated 9-11 months later. We conducted cognitive evaluation using spatial learning in a water maze and exploration behavior in an open field. Morphology of the brain was examined by MRI using diffusion-tensor imaging (DTI) and immunohistochemistry staining of the brain and eyes. Cognitive deficit was found in spatial memory and exploration behavior in both occluded groups. Brain and eyes histology presented severe damage in the bilateral group, compared to the unilateral one. DTI revealed an increase in mean diffusivity (MD) in the ventral thalamus and a decrease in fractional anisotropy in optic nerve and optic tract in bilateral rats, while unilateral rats showed only an increase in MD in the ventral pons. In those areas, a significant change in astrocytes, microglia, and number of apoptotic cells were found. Bilateral occlusion produced severe damage to both retinas, while unilateral occlusion produced damage mainly in the occluded side. We found that mild carotid stenosis, even in a unilateral occlusion, creates behavioral abnormalities presented by brain and eye histopathology. The results support our hypothesis that gradual formation of mild carotid stenosis along the life course leads to progressive damage that may create different degenerative diseases at a later age.

Correction: Procollagen C-Proteinase Enhancer 1 (PCPE-1) as a Plasma Marker of Muscle and Liver Fibrosis in Mice.

[This corrects the article DOI: 10.1371/journal.pone.0159606.].

Procollagen C-Proteinase Enhancer 1 (PCPE-1) as a Plasma Marker of Muscle and Liver Fibrosis in Mice.

Current non-invasive diagnostic methods of fibrosis are limited in their ability to identify early and intermediate stages of fibrosis and assess the efficacy of therapy. New biomarkers of fibrosis are therefore constantly sought for, leading us to evaluate procollagen C-proteinase enhancer 1 (PCPE-1), a fibrosis-related extracellular matrix glycoprotein, as a plasma marker of fibrosis. A sandwich ELISA that permitted accurate measurements of PCPE-1 concentrations in mouse plasma was established. Tissue fibrosis was assessed using histochemical, immunofluorescence, and immunoblotting analyses for type I collagen and PCPE-1. The normal plasma concentration of PCPE-1 in 6 weeks to 4 months old mice was ~200 ng/ml (189.5 ± 11.3 to 206.8 ± 13.8 ng/ml). PCPE-1 plasma concentrations in four and 8.5 months old mdx mice displaying fibrotic diaphragms increased 27 and 40% respectively relatively to age-matched control mice, an increase comparable to that of the N-propeptide of procollagen type III (PIIINP), a known blood marker of fibrosis. PCPE-1 plasma levels in mice with CCl4-induced liver fibrosis increased 34 to 50% relatively to respective controls and reflected the severity of the disease, namely increased gradually during the progression of fibrosis and went down to basal levels during recovery, in parallel to changes in the liver content of collagen I and PCPE-1. The results favor PCPE-1 as a potential new clinically valuable fibrosis biomarker.

S-Allylmercapto-N-acetylcysteine (ASSNAC) protects cultured nerve cells from oxidative stress and attenuates experimental autoimmune encephalomyelitis.

Oxidative stress and/or low cellular glutathione are associated with development and progression of neurodegenerative diseases. We have shown that S-allylmercapto-N-acetylcysteine (ASSNAC) up-regulates the level of glutathione and phase II detoxifying enzymes in cultured vascular endothelial cells. The present study demonstrates that exposure of nerve cell lines to ASSNAC significantly increases the cellular level of glutathione probably via activation of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and protects the cells from tBuOOH-induced cytotoxicity. Furthermore, ASSNAC increases the level of mice spinal cord and brain glutathione (by 54% and 47%, respectively) and attenuates the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in mice. In conclusion, these data implicate ASSNAC to protect nerve cells, both in vitro and in vivo, from oxidative stress and thereby to attenuate the clinical symptoms of EAE, suggesting its potential use for the treatment of neurodegenerative diseases.

Involvement of CD24 in angiogenesis in a mouse model of oxygen-induced retinopathy.

PURPOSE: To investigate a possible involvement of CD24 in vascular remodeling and angiogenesis in retinopathy of prematurity (ROP) in a mouse model of oxygen-induced retinopathy. MATERIALS AND METHODS: 17 CD24 knockout (KO) and 12 wild-type (WT) C57BL/6 mice were used. Group 1 mice were exposed to oxygen concentrations of 75 ± 2% from postnatal day (P) 7 to P12. Group 2 mice were raised in room air. At P17, all mice underwent fluorescein-conjugated-dextran perfusion and were sacrificed. The flat-mounted retinas were scored manually and digitally by a new computerized algorithm, according to blood vessel obliteration, tortuosity, vascular tufts and neovascularization formation. RESULTS: Fifty four retinal whole mounts were available for analysis and scoring. Group 1 retinas had significantly higher values of vaso-obliteration, tufts, neovascularization, vessel tortuosity and higher mean retinopathy scores than Group 2 retinas (KO mice: 9.0 ± 0.27 vs. 0.74 ± 0.2, respectively, P < 0.0001; WT mice: 7.58 ± 0.40 vs. 1.17 ± 0.27, respectively, P < 0.0001). Manual scoring in Group 1 revealed higher values of neovascularization, tortuosity and mean retinopathy scores in KO mice vs. WT mice (9.0 ± 0.27 vs. 7.58 ± 0.40, respectively, P = 0.009). Digital scoring revealed a higher neovascularization score in KO mice as well (13.72 ± 0.82% vs. 8.06 ± 0.27%, P < 0.0001). All mice had similar vaso-obliteration areas. There were no significant differences between KO and WT mice in Group 2. CONCLUSIONS: Absence of CD24 may have a deleterious effect on angiogenesis occurring in the second stage of ROP development, though its role in vessel obliteration during the first stage of ROP is probably limited.

Allicin up-regulates cellular glutathione level in vascular endothelial cells.

BACKGROUND: Allicin in garlic is the primary active compound known to rapidly interact with free thiols. AIMS OF THE STUDY: To examine the effect of allicin on gene expression and glutathione cellular level in vascular endothelial cells. METHODS: Cultured endothelial cells were exposed to allicin; mRNA was prepared and subjected to Micro-array and Real-Time PCR. Glutathione cellular level was determined on cell lysates. RESULTS: Micro-array analysis demonstrated allicin-induced up- and down-regulation of 116 and 100 genes, respectively. Up-regulated genes included the phase II detoxifying enzymes thioredoxin reductase 1 and 2, heme oxygenase-1 and glutamate cysteine lygaze modifier subunit, the rate limiting enzyme in glutathione biosynthesis. Endothelial cells exposed to allicin and its derivatives containing glutathione or cysteine residues increased cellular glutathione. Allicin increased the glutathione level in a concentration and time-dependent manner up to 8-fold at a concentration of 10-20 microM after 28 h exposure. Furthermore, allicin derivative-treated cultures demonstrated a 50% decrease in tBuOOH cytotoxicity. CONCLUSIONS: These results may suggest a putative role for allicin and its derivatives in preventing reactive oxygen species damage by up-regulating the phase II detoxifying enzymes and increasing the cellular glutathione level.

Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells.

Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue.