Expression and activity of catalase in the nervous tissue of oxaliplatin-treated animals.

<p>On day 21, dorsal root ganglia (DRG) and spinal cord were analyzed to measure both expression and activity of catalase. Densitometric analysis and representative Western blot of catalase expression in DRG (A) and spinal cord (C) are shown. GAPDH normalization was performed for each sample. Catalase enzymatic activity measurements in DRG (B) and spinal cord (D). Values are expressed as the mean ± S.E.M. percent of control of 10 rats per group, performed in 2 different experimental set. Each value represents the mean of *P<0.05 vs vehicle + vehicle; ∧P<0.05 vs oxaliplatin + vehicle.</p

Expression and activity of catalase in astrocyte cell culture.

<p>Astrocytes (5·10⁵ cells/well) were treated with the PPARγ antagonist G3335 (30 µM) or with oxaliplatin (1 µM) in the absence or in the presence of the PPARγ agonist rosiglitazone (10 µM). Expression and activity were measured after 48 h- (A and B, respectively) or 5 day-treatment (C and D, respectively). GAPDH normalization was performed for each sample. Values are expressed as the mean ± S.E.M. percent of control of three experiments. Control condition was arbitrarily set as 100%. *P<0.05 vs control; ∧P<0.05 vs 1 µM oxaliplatin.</p

Levels of carbonylated proteins in the spinal cord of oxaliplatin-treated rats.

<p>At 21^th day, the lumbar tract of the spinal cord was explanted and analyzed to measure protein oxidation. Densitometric analysis (top panel) and representative Western blot (lower panel) are shown. β-actin normalization was performed for each sample. Values are expressed as the mean ± S.E.M. percent of control of 10 rats per group, performed in 2 different experimental set. Each value represents the mean of *P<0.05 vs vehicle + vehicle; ∧P<0.05 vs oxaliplatin + vehicle.</p

Pain threshold measurements.

<p>A) Noxious stimulus, Paw-pressure test. Rats were daily intraperitoneally treated with 2.4 mg kg⁻¹ oxaliplatin (dissolved in 5% glucose). Rosiglitazone (3 and 10 mg kg⁻¹, suspended in CMC) was per os daily administered starting from the first day of oxaliplatin administration. B) Non-noxious stimulus, Cold plate test. The response to a thermal stimulus was evaluated by cold plate test measuring the latency (seconds) to pain-related behaviors (lifting or licking of the paw). Control animals were treated with vehicles. Behavioral measures were performed on day 7, 14 and 21, 24 h after the last treatment. Each value represents the mean of 10 rats per group, performed in 2 different experimental set. *P<0.01 vs vehicle + vehicle (control); ∧P<0.01 vs oxaliplatin + vehicle.</p

Lipid peroxidation in spinal cord.

<p>On day 21, the lumbar tract of the spinal cord was explanted for the analysis of lipid peroxidation. Data were expressed as mean ± SEM of Thiobarbituric Acid Reactive Substances (TBARS) levels (µmol/mg protein). Each value represents the mean of 10 rats per group, performed in 2 different experimental set. *P<0.01 vs vehicle + vehicle (control); ∧P<0.05 vs oxaliplatin + vehicle.</p

HT-29 cell viability, 48 h.

<p>Ht-29 cells were treated with increasing concentrations of oxaliplatin (1–300 µM) in the presence or in the absence of 10 µM rosiglitazone. Incubation was allowed for 48 h. Cell viability was measured by MTT assay. Control condition was arbitrarily set as 100% and values are expressed as the mean ± S.E.M. of three experiments. *P<0.05 and **P<0.01 in comparison to control (oxaliplatin 0 µM).</p

Motor coordination in oxaliplatin-treated rats.

<p>The integrity of the animals’ motor coordination was assessed using a rota-rod apparatus. Rats were placed on a rotating rod (10 rpm) for a maximum of 10 minutes (600 seconds). The number of falls (A) and the time spent in the balance (B) during 10 minutes were counted. Treatments (oxaliplatin 2.4 mg kg⁻¹ i.p. and rosiglitazone 3 and 10 mg kg⁻¹ p.o.) were performed daily. Motor coordination was evaluated on day 21, 24 h after the last treatment. Each value represents the mean of 10 rats per group, performed in 2 different experimental set. *P<0.01 vs vehicle + vehicle (control); ∧P<0.05 vs oxaliplatin + vehicle.</p

OLE increases Beclin 1 and LC3 in the cortex of wt and TgCRND8 mice.

<p>Representative images of Beclin 1 (A) and LC3 (C) immunoreactivity showing an intense bright and punctate Beclin 1 staining in the soma, perikarya and dendrites of neurons and strong and bright LC3 puncta in the neuronal cell bodies and processes of neurons in the somatosensory/parietal cortex of Tg mice and, to a lesser extent, in the wt mice fed with OLE, as compared to age-matched untreated Tg and wt mice (n = 5/group). Scale bars = 50 µm applies for the low magnification images and 20 µm applies for the high magnification images of untreated and OLE-fed 6-month-old Tg mice. (B) and (D) Western blotting analysis of Beclin 1 (B) and LC3 (D) levels in cortical tissue, exemplified for mice of 3.5 months of age, normalized for β-actin, (n = 6–7/group). LC3 levels are expressed as LC3-II/LC3-I levels. In the cortex of OLE-fed animals Beclin 1 levels show a trend towards an increase in the wt mice and in the OLE-fed Tg mice Beclin 1 and LC3 levels were significantly increased respect to age-matched untreated wt and Tg mice. (**P<0.01). Data are reported as mean values ± S.E.M.</p

Autophagosome-lysosome fusion in the cortex of untreated and OLE-fed wt and Tg mice.

<p>(A) Merge of cathepsin B (green) and p62 (red) immunoreactivity in 3.5-month-old wt and Tg mice. Co-localization between cathepsin B and p62 staining was detected as bright yellow puncta in small-sized lysosomes in the cortex of untreated wt mice and OLE-fed Tg and wt mice (n = 6/group). Scale bar = 25 µm. Inset: high magnification of a p62 and cathepsin B positive neuron. Scale bar = 14 µm. (B) Western blotting analysis of cathepsin B and p62 levels in the cortex of 3.5-month-old wt and Tg mice; wt = pool of untreated and OLE-fed wt mice (n = 10); Tg = untreated and OLE-fed Tg mice (N = 6/group). Both cathepsin B and p62 levels were significantly increased in OLE-fed Tg mice, as compared to untreated Tg mice. Data are representative of four experiments and are normalized for β-actin and reported as mean values ± S.E.M. *P<0.05, ** P<0.01. (C) Single and double fluorescent immunohistochemistry with cathepsin B (green) and p62 (red) Abs in the cortex of untreated and OLE-fed Tg mice. In the untreated Tg mice, bright cathepsin B immunoreactivity occurred in enlarged lysosomal compartments (arrows), p62 immunoreactivity was light and no co-localization between cathepsin B and p62 was found. Inset: high magnification of a cell with cathepsin B-positive giant lysosomes. In the OLE-fed Tg mice, a bright cathepsin B immunoreactivity appeared in small-sized lysosomes, p62 immunoreactivity was greater than in untreated Tg mice and a significant co-localization between cathepsin B and p62 was evident. (n = 6). Scale bars = 25 µm applies for single cathepsin B staining and 10 µm applies to the inset and high magnification images.</p

Antibodies employed in the study.

<p>Legend: aa, amino acid; WB, western blot; IHC, immunohistochemistry, ND, not done.</p