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Urs), rats were sacrificed by i.p. injection of pentobarbital (120 mg/kg). Blood and cerebella were harvested immediately. Blood or serum was used to measure glucose, insulin, cholesterol, triglycerides, and free fatty acid levels, as previously described [45,46]. Cerebella were harvested for histopathological, biochemical, and molecular studies. For histopathology, tissue samples were immersion f
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Urs), rats were sacrificed by i.p. injection of pentobarbital (120 mg/kg). Blood and cerebella were harvested immediately. Blood or serum was used to measure glucose, insulin, cholesterol, triglycerides, and free fatty acid levels, as previously described [45,46]. Cerebella were harvested for histopathological, biochemical, and molecular studies. For histopathology, tissue samples were immersion f
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Antibody (1:10000) and Amplex Red soluble fluorophore [79]. Amplex Red fluorescence was measured (Ex 579/Em 595) in a SpectraMax M5 microplate reader (Molecular Devices Corp., Sunnyvale, CA). Negative control reactions included substitutions with nonrelevant primary or secondary antibodies, and omission of primary or secondary antibody. Immunoreactivities were normalized to protein content as dete
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S described elsewhere [79]. For molecular and biochemical assays, cerebella were snap-frozen in a dry ice-methanol bath and stored at -80 . We studied cerebellar tissue because the cerebellum: 1) requires intact insulin/IGF signaling to maintain its structural and functional integrity [80,81]; 2) is severely damaged by i.c.-STZ mediated neurodegeneration [19,22]; 3) although relatively spared, it
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Changes characterized by focal loss of Purkinje neurons (Fig. 1-A3). NDEA exposure, with or without chronic HFD feeding, resulted in loss of Purkinje cells (Figs. 1-A2, 1-A4) and variable thinning of the granule cell layer. Immunohistochemical staining demonstrated similar levels and distributions of GFAP immunoreactivity in cells distributed in the granule layer of control (Fig 1-B1) and HFD-fed
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N exist inTable 2 High Fat Diet Feeding and NDEA Treatment Cause Type 2 Diabetes MellitusAssay Body Wt (g) Glucose (mg/dL) Insulin (ng/ml) Leptin Adiponectin Triglyceride (mg/ml) Free Fatty Acids (mM/mg prot) Cholesterol (mg/ml) LFD+VEH 265.100 ?14.050 111.5 ?1.66 0.0611 ?0.017 4.649 ?0.789 20864 ?1454 0.399 ?0.028 0.150 ?0.003 0.943 ?0.024 LFD+NDEA 266.600 ?19.970 128.8* ?4.31 0.163* ?0.038 4.775
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Ysiol Cell Physiol. 2012;302(2):C383?91. 68. Sachdev U, Cui X, Hong G, Namkoong S, Karlsson JM, Baty CJ, et al. High mobility group box 1 promotes endothelial cell angiogenic behavior in vitro and improves muscle perfusion in vivo in response to ischemic injury. J Vasc Surg. 2012;55(1):180?1.Yang et al. Cell Bioscience (2015) 5:Page 10 of69. Kang R, Livesey KM, Zeh HJ, Loze MT, Tang D. HMGB1: a
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S performed using the ABC method, and revealed with DAB (brown precipitate)-see Experimental Procedures. Sections were lightly counterstained with Hematoxylin (blue) to help reveal the tissue architecture. Cerebellar layers: ml = molecular layer; pc = Purkinje cell layer; gc = granule cell layer; wm = white matter. Note focal pc loss in A2, and large zones of pc loss in A3 and A4. (Original Magnif