Effects of TNBS-induced colitis on colonic PK2L mRNA expression.

<p>Four days after TNBS instillation, an increase in colonic PK2L mRNA expression was revealed in control colitic rats (Fisher’s LSD <i>post-hoc</i> ***p<0.001 vs. control healthy rats). PK2L mRNA levels in CORT-nursed colitic animals were significantly lower (Fisher’s LSD <i>post-hoc</i> ***p<0.001 vs. control colitic). The colonic relative mRNA expression levels are expressed in relation to β-actin and presented as fold increase relative to control rats. (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4).</p

Effects of TNBS-induced colitis on colonic PKR2 mRNA and protein expression.

<p>(A) Four days after TNBS instillation, an increase in colonic PKR2 mRNA expression was observed in colitic rats (***p<0.001colitic vs. healthy group). The colonic relative mRNA expression levels are expressed in relation to β-actin and presented as fold increase relative to control rats. (B) Western blot analysis showed no differences between experimental groups. Results are expressed as the ratio of the optical density (OD) of the PKR2 and the β-actin band. (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4). A representative western blot image is shown.</p

Effects of TNBS-induced colitis on colonic phospho-p65NF-κB expression.

<p>Four days after TNBS instillation, an increase in colonic phospo-p65NF-κB expression was observed in colitic animals (***p<0.001 colitic vs. healthy group). A reduction in phospo-p65NF- κB was observed in CORT-nursed rats (**p<0.01 CORT-nursed vs. control group). Tissue sections were incubated with a primary antibody against phospho-p65NF-κB (red signal). Nuclei were visualised by Hoechst 33258 (blue signal). Results are expressed as percentage (%) of positive labelled with respect to the total area (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4). Representative immunofluorescence images are shown.</p

Effects of TNBS-induced colitis on colonic GR expression.

<p>Four days after TNBS instillation, increased colonic GR expression was observed in both control and CORT-nursed colitic animals (Fisher’s LSD <i>post-hoc</i>, **p<0.01, ***p<0.001 vs. own healthy animals). The CORT-nursed colitic group showed GR expression levels greater than those of the control colitic group (Fisher’s LSD <i>post-hoc</i>, *p<0.05). The results are expressed as the ratio of optical density (OD) of the GR to that of the β-actin band (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4). A representative western blot image was reported.</p

Effects of TNBS-induced colitis on colonic IL-1β and TNF-α mRNA expression.

<p>(A) An increase in colonic IL-1β mRNA expression was observed in control colitic rats (Fisher’s LSD <i>post-hoc</i>, ***p<0.001 vs. control healthy rats). A reduction in IL-1β mRNA expression was observed in CORT-nursed colitic rats (Fisher’s LSD <i>post-hoc</i>, ***p<0.001 vs. control colitic rats). (B) An increase in colonic TNF-α mRNA expression was observed in control colitic rats (Fisher’s LSD <i>post-hoc</i>, ***p<0.001 vs. control healthy rats). A reduction in TNF-α mRNA expression was observed in CORT-nursed colitic rats (Fisher’s LSD <i>post-hoc</i>, ***p<0.001 vs. control healthy rats). The colonic relative mRNA expression levels are expressed in relation to β-actin and presented as fold increases relative to control rats. (Control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4).</p

Effects of TNBS-induced colitis on colonic PKR1 mRNA and protein expression.

<p>(A) Four days after TNBS instillation, no significant differences were observed in colonic PKR1 mRNA expression between groups. The colonic relative mRNA expression levels are expressed in relation to β-actin and presented as fold increase relative to control rats. (B) Analysis of PKR1 protein expression showed no differences between experimental groups. Results are expressed as the ratio of the optical density (OD) of the PKR1 and the β-actin band. (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4). A representative western blot image is shown.</p

Immunofluorescence analysis of Kv1.4 subunit expression in hippocampus and cerebral cortex.

<p>Upper panel. Representative immunofluorescence photomicrographs showing Kv1.4 expression in <b>a</b>) hippocampus and <b>b</b>) frontal cortex after memory tests in the four experimental treatments: Control (Saline), Aβ_25–35-i.c.v. treated rats (Abeta), Aβ_25–35-i.c.v. and SP-i.p. treated rats (Abeta+SP), SP-i.p. treated rats (SP). Brain sections were labeled with the neuronal marker NeuN (green) and with the anti Kv1.4 antibody (red). As shown by the merge channel all neurons are Kv1.4 positive. Note the diffuse increase in Kv1.4 fluorescence intensity in the Abeta group and the decrease in the Abeta+SP group compared to the Control. Scale bar: a) 20 µm; b) 60 µm. Lower panel. Histograms showing image analysis performed on neuronal cytoplasm (first row) and the surrounding neuropil (second row). The indexes used were: total fluorescence intensity, vesicles diameters, and vesicles fluorescence intensity. Data represent means (±S.E.M.) obtained from three independent experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Tukey's test for multiple comparisons (**p<0.01 versus Saline; #p<0.05, ##p<0.01 versus Aβ_25–35treatment).</p

Western blot analysis of Kv1.4 subunit expression in hippocampus and cerebral cortex.

<p>Representative immunoblot of (<b>a</b>) hippocampus and (<b>b</b>) cerebral cortex enriched membrane proteins (50 µg/lane) from (Ctr), Aβ_25–35, Aβ_25–35+SP and SP treated rats. Protein markers are shown at right (in kDa). The immunoreactive signals at 97 and 110 kDa were quantified and normalized against β-actin and expressed as a percentage of the control (Ctr). Data represent mean (±SEM) from 5 independent experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Tukey's test for multiple comparisons (**p<0.01 versus Ctr value; #p<0.05 versus Aβ_25–35 treatment).</p

Neuroprotective effects of SP on memory impairments induced by intracerebroventricular injection of Aβ25–35.

<p>(a) Timeline and experimental design. All animals received an infusion (i.c.v.) of Aβ_25–35 (2 µg/µl; 10 µL injection volume) or its vehicle (PBS 10 µL injection volume) and daily treated (7 days) with SP (50 µg/ml/Kg, i.p.) or its vehicle (saline solution 0.9%, i.p.). On the 31^st day after surgery rats were given a daily training session of 4 trials for 3 consecutive days (days 31^st–33^rd). On the 34^th day after surgery the retention of the spatial training was assessed during a 1 min probe trial. On the 35^th day after surgery rats were given a daily training session of 5 trials for 4 consecutive days (days 35^th–38^th). (b) Mean (±S.E.M.) distance traveled to the escape platform on 4 trials of 3 consecutive days of acquisition learning sessions. (c) Time spent (mean ±S.E.M.) during the 1-minute probe trial in the target quadrant and (d) illustrative paths of all animals for the probe test session. (e) Mean (±S.E.M.) distance traveled to the escape platform on 4 trials of 4 consecutive days of the reversal learning sessions (the hidden platform were relocated in a new position each day). * p<0.05 Aβ_25–35/Sal <i>vs</i> PBS/Sal; # p<0.05 Aβ_25–35/Sal <i>vs</i> PBS/SP; $ p<0.05 Aβ_25–35/Sal <i>vs</i> Aβ_25–35/SP. PBS/Sal, n = 10; PBS/SP, n = 10; Aβ_25–35/Sal n = 12; Aβ_25–35/SP, n = 10.</p

SP reduced Aβ25–35-induced overexpression of Kv1.4 subunit in rat hippocampal neurons.

<p><b>a</b>) Example of Western blot obtained from hippocampal cultures exposed to 20 µM Aβ_25–35 (Aβ alone or in the presence of SP (100 nM) and analyzed 48 h later using a polyclonal antibody against Kv1.4 subunit. The same blots were stripped and reprobed with an antibody against β-actin as internal control (lower panels). Quantitative analysis is depicted below the blots and was determined by band densitometry analysis considering the values found in CTR cells as 100. Data represent means (±S.E.M.) obtained from 4 independent experiments run in duplicate. (**p<0.001 versus CTR, #p<0.05 versus Aβ_25–35 treatment). <b>b</b>) Representative immunofluorescence photomicrographs showing Kv1.4 expression in primary hippocampal cultures. Note the increase in immunofluorescence in the Aβ_25–35 neurons, as compared to control neurons, reversed by SP treatment. Images were obtained from three independent experiments. Scale bar: 20 µm.</p