Effects of SA4503 and DTG on microglia after Hypoxia/Aglycemia.

<p>(<b>A</b>) Iba1 expressing microglia 24 h after normoxic control or combined H/A. Scale bar 10 µm. (<b>B</b>) Nitrite concentrations in the supernatant medium 24 h after stimulation as indicated in the figure. SA4503 (SA, 10 µM) and DTG (300 µM) were added to the culture medium immediately after H/A or normoxic control stimulation, respectively. Stimulation with lipopolysaccharide (LPS, 1 µg/mL) served as control. Abbreviations: ctrl – normoxic control, H/A – Hypoxia/Aglycemia. Data are presented as means ± SEM, *p<0.01 vs all control stimulations, #p<0.05 vs H/A DTG, one-way ANOVA and posthoc Bonferroni correction. <b>(C)</b> Levels of TNF-α and IL-1β after control or H/A stimulation and respective treatments (SA4503, 10 µM; DTG, 300 µM) for 24 hours. Data are presented as means ± SEM, n = 20 each experimental condition, *p<0.01 vs all control stimulations, #p<0.05 vs H/A DTG, one-way ANOVA and posthoc Bonferroni correction.</p

Physiological parameters of rats subjected to tMCAO at the time of recirculation.

<p>Data are presented as means ± standard deviation. No statistical differences were observed between the treatment groups.</p

Analysis of cytokine in the ischemic hemipshere.

<p>Levels of intraparenchymal TNF-α, IFN-γ, IL-1β, IL-4, IL-5 and IL-13 of the ischemic core and peri-infarct region were measured from ischemic animals treated with saline (n = 8, white bars) or SA4503 (0.5 mg/kg, s.c., n = 8, gray bars) and of cortices from sham treated animals (saline n = 3; SA4503, 0.5 mg/kg s.c., n = 3) by multiplex ELISA using a SECTOR Imager 6000 reader. Data are presented as means ± sd. Statistical analysis was performed one-way ANOVA and posthoc Bonferroni correction (*<i>p</i><0.05 vs sham saline, **<i>p</i><0.05 vs ischemic core saline, #<i>p</i><0.05 vs sham SA4503, ##<i>p</i><0.05 vs ischemic core SA4503).</p

Effect of SA4503 on infarct volume and functional recovery after MCAO.

<p>(<b>A</b>) Infarct volume from vehicle (saline; n = 8) and SA4503 treated (0.5 mg/kg; n = 8) rats at 7 days following MCAO. Infarct volumes are calculated indirectly (nonlesioned contralateral hemisphere minus nonlesioned area of the ischemic hemisphere). No statistical difference was obtained between the treatment groups. (<b>B</b>) Evaluation of sensori-motor function in saline (vh, n = 10) and SA4503 treated (0.5 mg/kg, n = 12) rats at the indicated times before and after MCAO. Animals were tested on the rotating pole at 10 turns per minute to the left. Similar results were obtained with 10 turns to the right (data not shown). No statistical difference was obtained between the treatment groups at all time points. (<b>C</b>) Forelimb strength of the non-paralyzed and paralyzed forelimb was measured by a grip strength test meter one day before and at day 7 following MCAO (MCAO vehicle n = 10; MCAO SA4503 n = 12); vh – vehicle, SA – SA4503.</p

Expression of Sig-1R in OX-42⁺ cells in the ischemic hemisphere - Effect of SA4503 treatment.

<p>Sig-1R immunoreactivity in a vehicle (<b>A</b>) and SA4503 (<b>B</b>) treated animal after MCAO. In both animals Sig-1R (Cy3, red) is expressed in OX-42⁺ cells (Cy5, green) in the infarct core and adjacent peri-infarct area. Scale bars: 50 µm. (<b>C</b>) Western blot for the Sig-1R of samples from the ischemic infarct core from SA4503 (0.5 mg/kg s.c.) and vehicle treated rats and respective densitometric analyses (mean±SD, *p<0.05, t-test) at day 7 following MCAO. Each lane represents an individual animal and for densitometric analysis n = 8 rats were included for each treatment group.</p

PCNA and OX-42 in the infarct core after MCAO.

<p>Representative Western blots of samples from the ischemic infarct core from SA4503 (0.5 mg/kg; n = 4) and vehicle treated (n = 4) rats at day 7 following MCAO. For densitometric analysis (mean±SD) n = 8 rats were included for each treatment group. Each lane represents an individual animal.</p

Iba1 levels in the infarct core after MCAO.

<p>(A) Western blot of samples from the infarct core and the peri-infarct area from SA4503 (0.5 mg/kg s.c.; n = 7) and vehicle treated rats (n = 6) and respective densitometric analyses (mean±SD, *p<0.05, t-test) at day 7 following MCAO. Note that each lane represents an individual animal. (B) Average Iba1 immunoreactivity in the ischemic hemisphere of vehicle and SA4503 rats on day 7 after tMCAo. Average Cy3 fluorescence intensity is presented per pixel (0 to 255) within the indicated areas considered as immunopositive Iba1, respectively (I/pixel; mean±s.d., n = 3 per treatment).</p

Experimental design.

<p>Experimental design.</p